Upregulation of type III endothelial cell nitric oxide synthase by agents that disrupt actin cytoskeletal organization

ABSTRACT

A use for agents that disrupt actin cytoskeletal organization is provided. In the instant invention, agents that disrupt actin cytoskeletal organization are found to upregulate endothelial cell Nitric Oxide Synthase activity. As a result, agents that disrupt actin cytoskeletal organization are useful in treating or preventing conditions that result from the abnormally low expression and/or activity of endothelial cell Nitric Oxide Synthase. Such conditions include hypoxia-induced conditions. Subjects thought to benefit mostly from such treatments include nonhyperlipidemics and nonhypercholesterolemics, but not necessarily exclude hyperlipidemics and hypercholesterolemics.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/115,387 filed on Jul. 14, 1998, now U.S. Pat. No. 6,423,751 entitledUPREGULATION OF TYPE III ENDOTHELIAL CELL NITRIC OXIDE SYNTHASE BYAGENTS THAT DISRUPT ACTIN CYTOSKELETAL ORGANIZATION. The contents of theabove-identified applications are hereby expressly incorporated byreference.

GOVERNMENT SUPPORT

The work resulting in this invention was supported in part by NIH GrantNo. RO1-HL-52233. The U.S. Government may therefore be entitled tocertain rights in the invention

FIELD OF THE INVENTION

This invention relates to the use of agents that disrupt actincytoskeletal organization as upregulators of Type III endothelial cellNitric Oxide Synthase. Further, this invention relates to methods thatemploy agents that disrupt actin cytoskeletal organization to treatconditions that result from the abnormally low expression and/oractivity of endothelial cell Nitric Oxide Synthase in a subject.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) has been recognized as an unusual messenger moleculewith many physiologic roles, in the cardiovascular, neurologic andimmune systems (Griffith, TM et al., J Am Coll Cardiol, 1988,12:797-806). It mediates blood vessel relaxation, neurotransmission andpathogen suppression. NO is produced from the guanidino nitrogen ofL-arginine by NO Synthase (Moncada, S and Higgs, E A, Eur J Clin Invest,1991, 21(4):361-374). In mammals, at least three isoenzymes of NOSynthase have been identified. Two, expressed in neurons (nNOS) andendothelial cells (Type III-ecNOS), are calcium-dependent, whereas thethird is calcium-independent and is expressed by macrophages and othercells after induction with cytokines (Type I-iNOS) (Bredt, D S andSnyder, S H, Proc Natl Acad Sci USA, 1990, 87:682-685, Janssens, S P etal., J Biol Chem, 1992, 267:22964, Lyons, C R et al., J Biol Chem, 1992,267:6370-6374). The various physiological and pathological effects of NOcan be explained by its reactivity and different routes of formation andmetabolism.

Recent studies suggest that a loss of endothelial-derived NO activitymay contribute to the atherogenic process (O'Driscoll, G, et al.,Circulation, 1997, 95:1126-1131). For example, endothelial-derived NOinhibits several components of the atherogenic process includingmonocyte adhesion to the endothelial surface (Tsao, P S et al.,Circulation, 1994, 89:2176-2182), platelet aggregation (Radomski, M W,et al., Proc Natl Acad Sci USA, 1990, 87:5193-5197), vascular smoothmuscle cell proliferation (Garg, U C and Hassid, A, J Clin Invest, 1989,83:1774-1777), and vasoconstriction (Tanner, F C et al., Circulation,1991, 83:2012-2020). In addition, NO can prevent oxidative modificationof low-density lipoprotein (LDL) which is a major contributor toatherosclerosis, particularly in its oxidized form (Cox, D A and Cohen,M L, Pharm Rev, 1996, 48:3-19).

It has been shown in the prior art that hypoxia downregulates ecNOSexpression and/or activity via decreases in both ecNOS genetranscription and mRNA stability (Liao, J K et al., J Clin Invest, 1995,96:2661-2666, Shaul, P W et al., Am J Physiol, 1997, 272: L1005-L1012).Thus, ischemia-induced hypoxia may produce deleterious effects, in part,through decreases in ecNOS activity.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is themicrosomal enzyme that catalyzes the rate limiting reaction incholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductaseinhibitor inhibits HMG-CoA reductase, and as a result inhibits thesynthesis of cholesterol. A number of HMG-CoA reductase inhibitors hasbeen used to treat individuals with hypercholesterolemia. Clinicaltrials with such compounds have shown great reductions of cholesterollevels in hypercholesterolemic patients. Moreover, it has been shownthat a reduction in serum cholesterol levels is correlated with improvedendothelium-dependent relaxations in atherosclerotic vessels (Treasure,CB et al., N Engl J Med, 1995, 332:481-487). Indeed, one of the earliestrecognizable benefits after treatment with HMG-CoA reductase inhibitorsis the restoration of endothelium-dependent relaxations or ecNOSactivity (supra, Anderson, T J et al., N Engl J Med, 1995, 332:488-493).

Although the mechanism by which HMG-CoA reductase inhibitors restoreendothelial function is primarily attributed to the inhibition ofhepatic HMG-CoA reductase and the subsequent lowering of serumcholesterol levels, little is known on whether inhibition of endothelialHMG-CoA reductase has additional beneficial effects on endothelialfunction.

By inhibiting L-mevalonate synthesis, HMG-CoA reductase inhibitors alsoprevent the synthesis of other important isoprenoid intermediates of thecholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP)and geranylgeranylpyrophosphate (GGPP) (Goldstein, J L and Brown, M S,Nature, 1990, 343:425-430). The isoprenoids are important lipidattachments for the post-translational modification of variety ofproteins, including G-protein and G-protein subunits, Heme-a, nuclearlamins, Ras, and Ras-like proteins, such as Rho, Rab, Rac, Ral or Rap(Goldstein, J L and Brown, M S, supra; Casey, P J, Science, 1995,268:221-225). The role that isoprenoids play in regulating ecNOSexpression, however, is not known.

Pulmonary hypertension is a major cause of morbidity and mortality inindividuals exposed to hypoxic conditions (Scherrer, U et al., N Engl JMed, 1996, 334:624-629). Recent studies demonstrate that pulmonaryarterial vessels from patients with pulmonary hypertension have impairedrelease of NO (Giaid, A and Saleh, D, N Engl J Med, 1995, 333:214-221,Shaul, P W, Am J Physiol, 1997, 272: L1005-L1012). Additionally,individuals with pulmonary hypertension demonstrate reduced levels ofecNOS expression in their pulmonary vessels and benefit clinically frominhalation nitric oxide therapy (Roberts, J D et al., N Engl J Med,1997, 336:605-610, Kouyoumdjian, C et al., J Clin Invest, 1994,94:578-584). Conversely, mutant mice lacking ecNOS gene or newborn lambstreated with the ecNOS inhibitor, Nw-monomethyl-L-arginine (LNMA),develop progressive elevation of pulmonary arterial pressures andresistance (Steudel, W et al., Circ Res, 1997, 81:34-41, Fineman, J R etal., J Clin Invest, 1994, 93:2675-2683). It has also been shown in theprior art that hypoxia causes pulmonary vasoconstriction via inhibitionof endothelial cell nitric oxide synthase (ecNOS) expression andactivity (Adnot, S et al., J Clin Invest, 1991, 87:155-162, Liao, J K etal., J Clin Invest, 1995, 96, 2661-2666). Hence, hypoxia-mediateddownregulation of ecNOS may lead to the vasoconstrictive and structuralchanges associated with pulmonary hypertension.

Often cited as the third most frequent cause of death in the developedcountries, stroke has been defined as the abrupt impairment of brainfunction caused by a variety of pathologic changes involving one orseveral intracranial or extracranial blood vessels. Approximately 80% ofall strokes are ischemic strokes, resulting from restricted blood flow.Mutant mice lacking the gene for ecNOS are hypertensive (Huang, P L etal., Nature, 1995, 377:239-242, Steudel, W et al., Circ Res, 1997,81:34-41) and develop greater intimal smooth muscle proliferation inresponse to cuff injury. Furthermore, occlusion of the middle cerebralartery results in 21% greater infarct size in “ecNOS knockout” micecompared to wildtype mice (Huang, Z et al., J Cereb Blood Flow Metab,1996, 16:981-987). These findings suggest that the ecNOS production mayplay a role in cerebral infarct formation and sizes. Additionally, sincemost patients with ischemic strokes have average or normal cholesterollevels, little is known on what the potential benefits of HMG-CoAreductase inhibitor administration would be in cerebrovascular events.

There exists a need to identify agents that improve endothelial cellfunction.

There also exists a need to identify agents that can be used acutely orin a prophylactic manner to treat conditions that result from low levelsof endothelial cell Nitric Oxide Synthase.

SUMMARY OF THE INVENTION

The invention involves the discovery that agents which disrupt actincytoskeletal organization can upregulate endothelial cell Nitric OxideSynthase (Type III) expression. The invention, therefore, is usefulwhenever it is desirable to restore endothelial cell Nitric OxideSynthase activity or increase such activity in a cell, tissue orsubject, provided the cell or the tissue expresses endothelial cellNitric Oxide Synthase.

Nitric Oxide Synthase activity is involved in many conditions, includingimpotence, heart failure, gastric and esophageal motility disorders,kidney disorders such as kidney hypertension and progressive renaldisease, insulin deficiency, etc. Individuals with such conditions wouldbenefit from increased endothelial cell Nitric Oxide Synthase activity.It also was known that individuals with pulmonary hypertensiondemonstrate reduced levels of Nitric Oxide Synthase expression in theirpulmonary vessels and benefit clinically from inhalation of NitricOxide. The invention therefore is particularly useful for treatingpulmonary hypertension. It also has been demonstrated that hypoxiacauses an inhibition of endothelial cell Nitric Oxide Synthase activity.The invention therefore is useful for treating subjects withhypoxia-induced conditions. It also has been discovered, surprisingly,that agents which disrupt actin cytoskeletal organization are useful forreducing brain injury that occurs following a stroke.

According to one aspect of the invention, a method is provided forincreasing endothelial cell Nitric Oxide Synthase activity in a subjectwho would benefit from increased endothelial cell Nitric Oxide Synthaseactivity in a tissue. The method involves administering to a subject inneed of such treatment an agent that disrupts actin cytoskeletalorganization in an amount(s) effective to increase endothelial cellNitric Oxide Synthase activity in the tissue of the subject, providedthat the agent that disrupts actin cytoskeletal organization is not arho GTPase function inhibitor. In one important embodiment agents thatdisrupt actin cytoskeletal organization do not affect cholesterol levelsin a subject. In certain embodiments, however, agents that disrupt actincytoskeletal organization as well as increasing endothelial cell NitricOxide Synthase activity in the tissue of a subject can also affectcholesterol levels in the subject. In certain embodiments, the subjectis nonhyperlipidimic. In other embodiments the amount is sufficient toincrease endothelial cell Nitric Oxide Synthase activity above normalbaseline levels established by age-controlled groups, described ingreater detail below.

The subject can have a condition characterized by an abnormally lowlevel of endothelial cell Nitric Oxide Synthase activity which ishypoxia-induced. In other embodiments the subject can have a conditioncomprising an abnormally low level of endothelial cell Nitric OxideSynthase activity which is chemically induced. In still otherembodiments the subject can have a condition comprising an abnormallylow level of endothelial cell Nitric Oxide Synthase activity which iscytokine induced. In certain important embodiments, the subject haspulmonary hypertension or an abnormally elevated risk of pulmonaryhypertension. In other important embodiments, the subject hasexperienced an ischemic stroke or has an abnormally elevated risk of anischemic stroke. In still other important embodiments, the subject hasheart failure or progressive renal disease. In yet other importantembodiments, the subject is chronically exposed to hypoxic conditions.

According to any of the foregoing embodiments, the preferred agent thatdisrupts actin cytoskeletal organization is selected from the groupconsisting of a myosin light chain kinase inhibitor, a myosin lightchain phosphatase, a protein kinase N inhibitor, a phospatidylinositol4-phosphate 5-kinase inhibitor, and cytochalasin D. In some embodimentsthe myosin light chain kinase inhibitor is selected from the groupconsisting of 2,3-butanedione 2-monoxime,1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydro-chloride. Likewise, in any of the foregoing embodiments, themethod can further comprise co-administering an endothelial cell NitricOxide Synthase substrate and/or co-administering an agent other than anagent that disrupts actin cytoskeletal organization that also increasesendothelial cell Nitric Oxide Synthase activity, and/or co-administeringat least one different agent that disrupts actin cytoskeletalorganization. A preferred agent other than an agent that disrupts actincytoskeletal organization is selected from the group consisting ofestrogens and angiotensin-converting enzyme (ACE) inhibitors. The agentsmay be administered to a subject who has a condition or prophylacticallyto a subject who has a risk, and more preferably, an abnormally elevatedrisk, of developing a condition. The inhibitors also may be administeredacutely.

According to another aspect of the invention, a method is provided forincreasing endothelial cell Nitric Oxide Synthase activity in a subjectto treat a condition favorably affected by an increase in endothelialcell Nitric Oxide Synthase activity in a tissue. Such conditions areexemplified above. The method involves administering to a subject inneed of such treatment an agent that disrupts actin cytoskeletalorganization in an amount effective to increase endothelial cell NitricOxide Synthase activity in the tissue of the subject, provided that theagent that disrupts actin cytoskeletal organization is not a rho GTPasefunction inhibitor. In important embodiments, agents that disrupt actincytoskeletal organization do not affect cholesterol levels in a subject.In certain embodiments, however, agents that disrupt actin cytoskeletalorganization as well as increase endothelial cell Nitric Oxide Synthaseactivity in the tissue of a subject can also affect cholesterol levelsin the subject. In certain embodiments, the subject isnonhyperlipidimic. Important conditions are as described above. Also asdescribed above, the method can involve co-administration of substratesof endothelial cell Nitric Oxide Synthase and/or co-administering anagent other than an agent that disrupts actin cytoskeletal organizationthat also increases endothelial cell Nitric Oxide Synthase activity,and/or co-administering at least one different agent that disrupts actincytoskeletal organization. Preferred compounds are as described above.As above, the agents that disrupt actin cytoskeletal organization withor without the co-administered compounds can be administered, interalia, acutely or prophylactically.

According to another aspect of the invention, a method is provided forreducing brain injury resulting from stroke. The method involvesadministering to a subject having an abnormally high risk of an ischemicstroke an agent that disrupts actin cytoskeletal organization in anamount effective to increase endothelial cell Nitric Oxide Synthaseactivity in the brain of the subject, provided that the agent thatdisrupts actin cytoskeletal organization is not a rho GTPase functioninhibitor. As above, important embodiments include the agent beingselected from the group consisting of a myosin light chain kinaseinhibitor, a myosin light chain phosphatase, a protein kinase Ninhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, andcytochalasin D. As above, in some embodiments a myosin light chainkinase inhibitor is selected from the group consisting of2,3-butanedione 2-monoxime,1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydro-chloride. Also as above, important embodiments includeco-administering a substrate of endothelial cell Nitric Oxide Synthaseand/or co-administering an agent other than an agent that disrupts actincytoskeletal organization that also increases endothelial cell NitricOxide Synthase activity, and/or co-administering at least one differentagent that disrupts actin cytoskeletal organization. Likewise, importantembodiments include prophylactic and acute administration of theagent(s).

According to another aspect of the invention, a method is provided fortreating pulmonary hypertension. The method involves administering to asubject in need of such treatment an agent that disrupts actincytoskeletal organization in an amount effective to increase pulmonaryendothelial cell Nitric Oxide Synthase activity in the subject, providedthat the agent that disrupts actin cytoskeletal organization is not arho GTPase function inhibitor. Particularly important embodiments are asdescribed above in connection with the methods for treating braininjury. Another important embodiment is administering the agentprophylactically to a subject who has an abnormally elevated risk ofdeveloping pulmonary hypertension, including subjects that arechronically exposed to hypoxic conditions.

According to another aspect of the invention, a method for treatingheart failure is provided. The method involves administering to asubject in need of such treatment an agent that disrupts actincytoskeletal organization in an amount effective to increase vascularendothelial cell Nitric Oxide Synthase activity in the subject, providedthat the agent that disrupts actin cytoskeletal organization is not arho GTPase function inhibitor. As discussed above, important embodimentsinclude prophylactic and acute administration of the agent(s). Preferredcompounds and co-administration schemes are as described above.

According to yet another aspect of the invention, a method is providedfor treating progressive renal disease. The method involvesadministering to a subject in need of such treatment an agent thatdisrupts actin cytoskeletal organization in an amount effective toincrease renal endothelial cell Nitric Oxide Synthase activity in thekidney of the subject, provided that the agent that disrupts actincytoskeletal organization is not a rho GTPase function inhibitor.Important embodiments and preferred compounds and schemes ofco-administration are as described above in connection with heartfailure.

According to another aspect of the invention, a method for increasingblood flow in a tissue of a subject is provided. The method involvesadministering to a subject in need of such treatment a first agent thatdisrupts actin cytoskeletal organization in an amount effective toincrease endothelial cell Nitric Oxide Synthase activity in the tissueof the subject, provided that the first agent is not an agent selectedfrom the group consisting of a rho GTPase function inhibitor andfasudil. In certain embodiments the first agent is not a myosin lightchain kinase inhibitor. In other embodiments the first agent is selectedfrom the group consisting of a myosin light chain phosphatase, a proteinkinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinaseinhibitor, and cytochalasin D. Other important embodiments includeco-administering a second agent to the subject with a conditiontreatable by the second agent in an amount effective to treat thecondition, whereby the delivery of the second agent to a tissue of thesubject is enhanced as a result of the increased blood flow. In certainembodiments where a second agent is administered, the conditiontreatable by the second agent does not involve the brain tissue.

The invention also involves the use of agents that disrupt actincytoskeletal organization in the manufacture of medicaments for treatingthe above-noted conditions. Important conditions, compounds, etc. are asdescribed above. The invention further involves pharmaceuticalpreparations that are cocktails of agents that disrupt actincytoskeletal organization according to the invention [non-rho GTPasefunction inhibitor(s)]. In certain embodiments, however, the cocktailscan include a rho GTPase function inhibitor(s) that disrupts actincytoskeletal organization together with the non-rho GTPase functioninhibitor agent of the invention. The invention also involvespharmaceutical preparations that are cocktails of agents that disruptactin cytoskeletal organization together with agents other than agentsthat disrupt actin cytoskeletal organization that also increase ecNOSactivity in a cell.

The invention also involves methods for increasing ecNOS activity in acell by contacting the cell with an effective amount of an agent thatdisrupts actin cytoskeletal organization (excluding rho GTPase functioninhibitors), alone, or together with any of the agents co-administeredas described above, or as a cocktail as described above.

In any of the foregoing aspects of the invention the agent can be anon-fasudil agent that disrupts actin cytoskeletal organization.

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ecNOS activity (FIG. 1A) and expression in wild-type SV-129 miceaortas (FIG. 1B) with and without treatment with simvastatin for 14days.

FIG. 2. ecNOS mRNA expression in the infarcted, ipsolateral (I) andnot-infarcted, contralateral (C) forebrain hemispheres of SV-129 micewith and without treatment with simvastatin.

FIG. 3. Northern blots showing the effects of mevastatin alone (FIG. 3A)or in combination (FIG. 3B) with FPP or GGPP on eNOS (ecNOS)steady-state mRNA levels after 24 h.

FIG. 4. Western blots showing the effects of mevastatin alone or incombination with FPP or GGPP or LDL-cholesterol on eNOS (ecNOS) proteinlevels after 24 h.

FIG. 5. Western blots showing the effects of C3 transferase, mevastatin,or L-mevalonate on eNOS (ecNOS) protein levels after 24 h.

FIG. 6. Western blots showing eNOS (ecNOS) protein levels aftertransfection with insertless vector, pcDNA3 (C), c-myc-wildtype-RhoA(wt), and c-myc-N19RhoA (dominant-negative rhoA mutant).

FIG. 7. Effects of C3 transferase, FPP, GGPP, and CNF-1 onmevastatin-induced eNOS (ecNOS) activity as determined byLNMA-inhibitable nitrite production at 24 h.

FIG. 8. Immunoblots showing the concentration-dependent effects of MLCkinase inhibitor H-7 on ecNOS protein levels after 24 hours.

FIG. 9. Northern blots showing ecNOS expression of endothelial cellstreated with cytochalasin D at 24 hours.

FIG. 10. Immunoblots showing the concentration-dependent effects of 2,3-butanedione 2-monoxime on ecNOS protein levels.

FIG. 11. Northern blots showing ecNOS expression of endothelial cellstreated with nocodazole for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

The invention is useful whenever it is desirable to increase endothelialcell Nitric Oxide Synthase (Type III isoform) activity in a cell, in atissue, or in a subject. A subject as used herein includes humans, nonhuman primates, dogs, cats, sheep, goats, cows, pigs, horses androdents. The invention thus is useful for therapeutic purposes and alsois useful for research purposes such as in testing in animal or in vitromodels of medical, physiological or metabolic pathways or conditions.Nitric Oxide Synthase is the enzyme that catalyzes the reaction thatproduces nitric oxide from the substrate L-arginine. As the nameimplies, endothelial cell nitric oxide Synthase refers to the Type IIIisoform of the enzyme found in the endothelium.

By “ecNOS activity”, it is meant the ability of a cell to generatenitric oxide from the substrate L-arginine. Increased ecNOS activity canbe accomplished in a number of different ways. For example, an increasein the amount of ecNOS protein or an increase in the activity of theprotein (while maintaining a constant level of the protein) can resultin increased “activity”. An increase in the amount of protein availablecan result from increased transcription of the ecNOS gene, increasedstability of the ecNOS mRNA or a decrease in ecNOS protein degradation.(The term “expression” is used interchangeably with the term “activity”throughout this application).

The ecNOS activity in a cell or in a tissue can be measured in a varietyof different ways. A direct measure would be to measure the amount ofecNOS present. Another direct measure would be to measure the amount ofconversion of arginine to citrulline by ecNOS or the amount ofgeneration of nitric oxide by ecNOS under particular conditions, such asthe physiologic conditions of the tissue. The ecNOS activity also can bemeasured more indirectly, for example by measuring mRNA half-life (anupstream indicator) or by a phenotypic response to the presence ofnitric oxide (a downstream indicator). One phenotypic measurementemployed in the art is detecting endothelial dependent relaxation inresponse to a acetylcholine, which response is affected by ecNOSactivity. The level of nitric oxide present in a sample can be measuredusing a nitric oxide meter. All of the foregoing techniques are wellknown to those of ordinary skill in the art, and some are described inthe examples below.

The present invention, by causing an increase in ecNOS activity, permitsnot only the re-establishment of normal base-line levels of ecNOSactivity, but also allows increasing such activity above normalbase-line levels. Normal base-line levels are the amounts of activity ina normal control group, controlled for age and having no symptoms whichwould indicate alteration of endothelial cell Nitric Oxide Synthaseactivity (such as hypoxic conditions, hyperlipidemia and the like). Theactual level then will depend upon the particular age group selected andthe particular measure employed to assay activity. Specific examples ofvarious measures are provided below. In abnormal circumstances, e.g.hypoxic conditions, pulmonary hypertension, etc., endothelial cellNitric Oxide Synthase activity is depressed below normal levels.Surprisingly, when using agents that disrupt actin cytoskeletalorganization according to the invention, not only can normal base-linelevels be restored in such abnormal conditions, but endothelial cellNitric Oxide Synthase activity can be increased desirably far abovenormal base-line levels of endothelial cell Nitric Oxide Synthaseactivity. Thus, “increasing activity” means any increase in endothelialcell Nitric Oxide Synthase activity in the subject resulting from thetreatment with agents that disrupt actin cytoskeletal organizationaccording to the invention, including, but not limited to, such activityas would be sufficient to restore normal base-line levels and suchactivity as would be sufficient to elevate the activity above normalbase-line levels.

As mentioned above, Nitric Oxide Synthase activity is involved in manyconditions, including stroke, pulmonary hypertension, impotence, heartfailure, gastric and esophageal motility disorders, kidney disorderssuch as kidney hypertension and progressive renal disease, insulindeficiency, hypoxia-induced conditions, etc. In one embodiment of theinvention the decrease in endothelial cell Nitric Oxide Synthaseactivity is cytokine induced. Cytokines are soluble polypeptidesproduced by a wide variety of cells that control gene activation andcell surface molecule expression. They play an essential role in thedevelopment of the immune system and thus in the development of animmune response. However, besides their numerous beneficial properties,they have also been implicated in the mechanisms for the development ofa variety of inflammatory diseases. For example, the cytokines TNF-a andIL-1 are thought to be part of the disease causing mechanism ofnon-cholesterol induced atherosclerosis, transplant arterial sclerosis,rheumatoid arthritis, lupus, scleroderma, emphysema, etc. Subjects ofsuch disorders exhibit lower levels of endothelial cell Nitric OxideSynthase activity (which is thus “cytokine induced”), and may benefitfrom therapy using the agents of the instant invention.

One important embodiment of the invention is treatment of ischemicstroke. Ischemic stroke (ischemic cerebral infarction) is an acuteneurologic injury that results from a decrease in the blood flowinvolving the blood vessels of the brain. Ischemic stroke is dividedinto two broad categories, thrombotic and embolic.

A surprising finding was made in connection with the treatment ofischemic stroke. In particular, it was discovered that treatmentaccording to the invention can reduce the brain injury that follows anischemic stroke. Brain injury reduction, as demonstrated in the examplesbelow, can be measured by determining a reduction in infarct size in thetreated versus the control groups. Likewise, functional tests measuringneurological deficits provided further evidence of reduction in braininjury in the treated animals versus the controls. Cerebral blood flowalso was better in the treated animals versus the controls. Thus, in thevarious accepted models of brain injury following stroke, a positiveeffect was observed in the treated animals versus the control animals.It is believed that all of the foregoing positive results areattributable to the upregulation of endothelial cell Nitric OxideSynthase activity, which is believed demonstrated in the examples below.

An important embodiment of the invention is treatment of a subject withan abnormally elevated risk of an ischemic stroke. As used herein,subjects having an abnormally elevated risk of an ischemic stroke are acategory determined according to conventional medical practice. Thiscategory includes, for example, subjects which are having electedvascular surgery. Typically, the risk factors associated with cardiacdisease are the same as are associated with stroke. The primary riskfactors include hypertension, hypercholesterolemia, and smoking. Inaddition, atrial fibrillation or recent myocardial infarction areimportant risk factors.

The treatment of stroke can be for patients who have experienced astroke or can be a prophylactic treatment. If prophylactic, then thetreatment is for subjects having an abnormally elevated risk of anischemic stroke, as described above. If the subject has experienced astroke, then the treatment can include acute treatment. Acute treatmentmeans administration of the agents that disrupt actin cytoskeletalorganization at the onset of symptoms of the condition or at the onsetof a substantial change in the symptoms of an existing condition.

Another important embodiment of the invention is treatment of pulmonaryhypertension. Pulmonary hypertension is a disease characterized byincreased pulmonary arterial pressure and pulmonary vascular resistance.Hypoxemia, hypocapnia, and an abnormal diffusing capacity for carbonmonoxide are almost invariable findings of the disease. Additionally,according to the present invention, patients with pulmonary hypertensionalso have reduced levels of ecNOS expression and/or activity in theirpulmonary vessels. Traditionally, the criteria for subjects with, or atrisk for pulmonary hypertension are defined on the basis of clinical andhistological characteristics according to Heath and Edwards(Circulation, 1958, 18:533-547).

Subjects may be treated prophylactically to reduce the risk of pulmonaryhypertension or subjects with pulmonary hypertension may be treated longterm and/or acutely. If the treatment is prophylactic, then the subjectstreated are those with an abnormally elevated risk of pulmonaryhypertension. A subject with an abnormally elevated risk of pulmonaryhypertension is a subject with chronic exposure to hypoxic conditions, asubject with sustained vasoconstriction, a subject with multiplepulmonary emboli, a subject with cardiomegaly and/or a subject with afamily history of pulmonary hypertension.

Another important embodiment of the invention involves treatinghypoxia-induced conditions. Hypoxia as used herein is defined as thedecrease below normal levels of oxygen in a tissue. Hypoxia can resultfrom a variety of circumstances, but most frequently results fromimpaired lung function. Impaired lung function can be caused byemphysema, cigarette smoking, chronic bronchitis, asthma, infectiousagents, pneumonitis (infectious or chemical), lupus, rheumatoidarthritis, inherited disorders such as cystic fibrosis, obesity,α₁-antitrypsin deficiency and the like. It also can result from non-lungimpairments such as from living at very high altitudes. Hypoxia canresult in pulmonary vasoconstriction via inhibition of ecNOS activity.

Another important embodiment of the invention is the treatment of heartfailure. Heart failure is a clinical syndrome of diverse etiologieslinked by the common denominator of impaired heart pumping and ischaracterized by the failure of the heart to pump blood commensuratewith the requirements of the metabolizing tissues, or to do so only froman elevating filling pressure.

In certain aspects of the invention, agents that disrupt actincytoskeletal organization are administered to subjects that wouldbenefit from increased endothelial cell Nitric Oxide Synthase activity.The administration of one or more agents that disrupt actin cytoskeletalorganization is in an amount(s) effective to increase endothelial cellNitric Oxide Synthase activity in tissue of the subject, provided thatthe agent that disrupts actin cytoskeletal organization used is not arho GTPase function inhibitor (See later discussion). In certainembodiments, the subject is both nonhypercholesterolemic andnonhypertriglyceridemic, i.e., nonhyperlipidemic. Such subjects arethought to benefit mostly from the treatments of the invention, but thetreatments do not necessarily exclude hyperlipidemic andhypercholesterolemic subjects.

A nonhypercholesterolemic subject is one that does not fit the currentcriteria established for a hypercholesterolemic subject. Anonhypertriglyceridemic subject is one that does not fit the currentcriteria established for a hypertriglyceridemic subject (See, e.g.,Harrison's Principles of Experimental Medicine, 13th Edition,McGraw-Hill, Inc., N.Y.). Hypercholesterolemic subjects andhypertriglyceridemic subjects are associated with increased incidence ofpremature coronary heart disease. A hypercholesterolemic subject has anLDL level of >160 mg/dL or >130 mg/dL and at least two risk factorsselected from the group consisting of male gender, family history ofpremature coronary heart disease, cigarette smoking (more than 10 perday), hypertension, low HDL (<35 mg/dL), diabetes mellitus,hyperinsulinemia, abdominal obesity, high lipoprotein (a), and personalhistory of cerebrovascular disease or occlusive peripheral vasculardisease. A hypertriglyceridemic subject has a triglyceride (TG) levelof >250 mg/dL. Thus, a hyperlipidemic subject is defined as one whosecholesterol and triglyceride levels equal or exceed the limits set asdescribed above for both the hypercholesterolemic andhypertriglyceridemic subjects.

The invention involves treatment of the foregoing conditions usingagents that disrupt actin cytoskeletal organization. Actin comprises alarge proportion of the cytoplasmic proteins of many cells. Actin ispresent primarily in its globular form (G-actin), a single polypeptide375 amino acids long, and is associated with one molecule ofnon-covalently bound ATP. The terminal phosphate of the ATP ishydrolysed after the actin polymerizes to form actin filaments (fibrousactin or F-actin). Actin filaments consist of a tight-helix of uniformlyoriented actin monomers. They are polar structures, with twostructurally different ends, and form the “core” of the actincytoskeleton. An actin cytoskeleton is thus a three dimensionalstructure that results from the interaction between actin filaments andother molecules that associate with the actin filaments (e.g.,cross-linking proteins such as filamin). The actin cytoskeleton mediatesa variety of biological functions in all eukaryotic cells. In additionto providing a structural framework around which cell shape and polarityare defined, its dynamic properties provide the driving force for cellsto move and to divide.

According to the present invention, it has been discovered that agentswhich disrupt actin cytoskeletal organization control endothelial cellNitric Oxide Synthase activity. In particular, agents that disrupt actincytoskeletal organization upregulate endothelial cell Nitric OxideSynthase activity.

According to the present invention, “agents that disrupt actincytoskeletal organization” are compounds, natural or synthetic, thatinterfere with actin cytoskeletal organization. Typically such agentswill interfere, for example, with stress fiber formation (contractilebundles of actin filaments and myosin), and/or focal contact (oradhesion plaque) assembly and upregulate endothelial cell Nitric OxideSynthase activity. The effects of such agents in a cell or in a tissueon actin cytoskeletal organization can be measured according to any artrecognized method. For example, a direct measure would be to performphalloidin staining (Sigma) on intact cells. A person of ordinary skillin the art could then determine (and quantitate) the effects of theagents of the invention by examining, for example, the structure of thestained actin stress-fibers and comparing such structure with the onewhich is normal and characteristic of an untreated cell.

Agents that disrupt actin cytoskeletal organization can exert theireffects at different levels and thus comprise different categories ofagents useful for practicing the present invention. The differentcategories include agents from those that disrupt actin cytoskeletalorganization at the nucleic acid level to agents that disrupt actincytoskeletal organization at the protein level.

Agents that disrupt actin cytoskeletal organization at the nucleotidelevel include chemicals, antisense nucleic acids, antibodies, catalyticnucleic acids including ribozymes, and proteins which interfere with theexpression of a gene that encodes a polypeptide which is a component ofthe actin cytoskeleton. Such exemplary polypeptides include but are notlimited to actin, myosin, tropomyosin, troponin, titin, nebulin,α-actinin, myomesin, C protein, filamin, talin, vinculin, cappingprotein, fibronectin receptor, ezrin, radixin, moiesin and the like.

Agents that disrupt actin cytoskeletal organization at the protein levelinclude organic molecules that inhibit or alter the formation andorganization of the actin cytoskeleton by interfering (e.g., viaantibody binding, etc.) or altering (e.g., via post-translationalmodification) an individual component of the actin cytoskeleton.Specifically included are proteins, peptides and lipid derivatives.Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

In certain embodiments, agents that disrupt actin cytoskeletalorganization include myosin light chain kinase (MLCK-Ser/Thr kinases)inhibitors, myosin light chain phosphatase (MLCP) stimulators, proteinkinase N (PKN) inhibitors, phospatidylinositol 4-phosphate 5-kinase(PIP5K) inhibitors, and cytochalasin D. Exemplary myosin light chainkinase inhibitors include BDM [2,3-butanedione 2-monoxime], ML-7[1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride], ML-9[1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepinehydrochloride], wortmannin, H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride], Fasudil (HA1077)[Hexahydro-1-(5-isoquinolinesulphonyl)-1H-1,4-diazepine],W-7[N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide] and A-3[N-(6-Aminoethyl)-5-chloro-1-naphthalenesulfonamide]. In preferredembodiments, agents that disrupt actin cytoskeletal organization includeBDM, ML-7, H-7 and cytochalasin D. Exemplary PKN inhibitors include“dominant negative” PKN peptides and purine analogues such as6-thioguanine. Exemplary PIP5K inhibitors include “dominant negative”PIP5K peptides. Exemplary MLCP stimulators include nucleic acids thatencode functional MLCP proteins and peptides (i.e., maintain thephosphatase activity of MLCP) and that are overexpressed (via anexpression vector) in the cells of interest of a subject according tothe invention.using genetic approaches well known in the art.Cytochalasin D is a preferred agent of the invention that belongs to thefamily of mold metabolites called cytochalasins. Cytochalasin D isthought to exert its function as an agent that disrupts actincytoskeletal organization by affecting actin polymerization. Othermembers of the cytochalasin family share this property (e.g.,Cytochalasin B), and are thus useful according to the invention.

Examples of agents that disrupt actin cytoskeletal organization alsoinclude “dominant negative” polypeptides of the polypeptide componentsof the actin cytoskeleton, some of which are exemplified above. Adominant negative polypeptide is an inactive variant of a protein,which, by interacting with the cellular machinery, displaces an activeprotein from its interaction with the cellular machinery or competeswith the active protein, thereby reducing the effect of the activeprotein. For example, a dominant negative receptor which binds a ligandbut does not transmit a signal in response to binding of the ligand canreduce the biological effect of expression of the ligand. Likewise, adominant negative catalytically-inactive kinase which interacts normallywith target proteins but does not phosphorylate the target proteins canreduce phosphorylation of the target proteins in response to a cellularsignal. Similarly, a dominant negative transcription factor which bindsto a promoter site in the control region of a gene but does not increasegene transcription can reduce the effect of a normal transcriptionfactor by occupying promoter binding sites without increasingtranscription.

The end result of the application of or expression of a dominantnegative polypeptide is a reduction in function of active proteins. Oneof ordinary skill in the art can assess the potential for a dominantnegative variant of a protein, and using standard mutagenesis techniquesto create one or more dominant negative variant polypeptides. Forexample, given the teachings contained herein and in the art, one ofordinary skill in the art can modify the sequence of a polypeptide (orthe gene encoding a polypeptide) of an actin cytoskeletal component (asdescribed earlier, e.g., actin, myosin, filamin, etc.) by site-specificmutagenesis, scanning mutagenesis, partial gene deletion or truncation,and the like. See, e.g., U.S. Pat. No. 5,580,723 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, 1989. The skilled artisan then can test thepopulation of mutagenized polypeptides for diminution in a selectedactivity (e.g., impaired myosin light chain phosphorylation andupregulation of ecNOS activity) and/or for retention of such anactivity. Other similar methods for creating and testing dominantnegative variants of a protein will be apparent to one of ordinary skillin the art.

Other examples of agents that disrupt actin cytoskeletal organizationinclude polypeptides which bind to components of the actin cytoskeletonand to complexes of the components of the actin cytoskeleton and bindingpartners. The invention, therefore, embraces peptide binding agentswhich, for example, can be antibodies or fragments of antibodies havingthe ability to selectively bind to components of the actin cytoskeleton.Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology.

A rho GTPase is a small, membrane-bound, Ras-related GTP-binding proteinthat functions by binding and hydrolyzing GTP. Rho GTPases function asmolecular switches, cycling between an inactive GDP-bound conformationand an active GTP-bound conformation. According to the presentinvention, “rho GTPase function inhibitors” are compounds, natural orsynthetic, that inhibit the normal function and localization of rhoGTPases (i.e., impair GTP binding by rho GTPases) and upregulateendothelial cell Nitric Oxide Synthase activity. Such compounds caninhibit rho GTPase function at different levels and thus comprisedifferent categories of agents useful for practicing the presentinvention. The different categories include agents from those thatinhibit rho GTPases at the nucleic acid level to agents that inhibit rhoGTPases at the protein level.

Agents that inhibit rho GTPases at the nucleotide level includechemicals, antisense nucleic acids, antibodies, catalytic nucleic acidsincluding ribozymes, and proteins which repress expression of a rhoGTPase gene locus.

Agents that inhibit rho GTPases at the protein level include organicmolecules that alter the intrinsic GTPase activity of the rhoGTP-binding protein, organic molecules that inhibit GDP/GTP exchange,and organic molecules that inhibit or alter post-translationalmodifications of rho GTPases. Specifically included are proteins,peptides and lipid derivatives.

Examples of agents that inhibit or reduce the intrinsic GTPase activityof a rho GTP-binding protein include cyclosporin, and “dominantnegative” polypeptides of the rho GTPase. A dominant negativepolypeptide is as described previously.

Dominant negative rho GTPase proteins include variants in which aportion of the GTP catalytic site has been mutated or deleted to reduceor eliminate GTP binding. Other examples include rho GTPase variants inwhich the conserved CAAX motif at their carboxy-terminus has beenmutated or deleted to reduce or eliminate post-tranlationalmodification. (C, cysteine; A, aliphatic amino acid; X, any amino acid).One of ordinary skill in the art can readily prepare such modifications.Examples of dominant negative rho GTPase peptides are described in theExamples section and include N19RhoA and CAAXRhoA.

Other examples of agents that inhibit or reduce the intrinsic GTPaseactivity of a rho GTP-binding protein include polypeptides which bind torho GTPase polypeptides and to complexes of rho GTPase polypeptides andbinding partners. The invention, therefore, embraces peptide bindingagents which, for example, can be antibodies or fragments of antibodieshaving the ability to selectively bind to rho GTPase polypeptides.Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology.

Examples of agents that inhibit the GDP/GTP exchange include proteinsand peptides that inhibit GDP-dissociation such as Ly-GDI and RhoGDI-3.Preferably, using genetic approaches well known in the art, suchproteins and peptides can be overexpressed (via an expression vector) inthe cells of interest of a subject according to the invention.

Post-translational modifications of rho GTPases are important in thatthey are necessary for the proper attachment (and thus function) of therho GTPases to the cell membrane. If rho GTPase polypeptides cannot beproperly modified (or if they are overmodified), they accumulate in thecytosol and are rendered inactive. Examples of agents that inhibitpost-translational modifications of rho GTPases includegeranylgeranylation inhibitors and guanine nucleotide exchangeinhibitors.

Geranylgeranylation inhibitors are compounds (natural or synthetic) thatinterfere with the geranylgeranylation of rho GTPases, and includeproteins, peptides and lipid derivatives. Thus, geranylgeranylationinhibition of rho GTPases can occur either by preventinggeranylgeranyl-pyrophosphate synthesis, or by inhibiting the enzymegeranylgeranyl transferase (GGT) which attachesgeranylgeranyl-pyrophosphate to the CAAX motif of rho GTPases.Geranylgeranyl-pyrophosphate synthesis inhibition can be performed bypreventing or inhibiting the formation of any of the intermediates inthe geranylgeranyl-pyrophosphate synthesis pathway. Examples includemevalonate inhibitors, isopentenyl-pyrophosphate inhibitors,geranyl-pyrophosphate inhibitors, farnesyl-pyrophosphate inhibitors andgeranylgeranyl-pyrophosphate inhibitors. Examples of such compoundsinclude farnesyl-transferase inhibitors disclosed in U.S. Pat. Nos.5,705,686 and 5,602,098, inhibitors of geranylgeranyl-transferasedisclosed in U.S. Pat. No. 5,470,832, the disclosure of which isincorporated herein by reference, and a-hydroxyfarnesylphosphonic acid.Additional geranylgeranyl-transferase inhibitors include GGTI-298(Finder, J D et al., J Biol Chem, 1997, 272:13484-13488).

Guanine nucleotide exchange inhibitors are agents that alsopost-translationaly modify and inactivate rho GTPases. They includebacterial protein toxins that ADP-ribosylate or glucosylate rho GTPases,or compounds that inhibit rho GTPase-specific guanine nucleotideexchange factor (GEF). Preferred such agents according to the inventioninclude Clostridium botulinum C3 transferase. The C3 transferaseenzymatically catalyses the transfer of ADP from NADH to Asp-41 of rho,rendering the rho GTPase resistant to GTP/GDP exchange by the rhoGTPase-specific guanine nucleotide exchange factors (GEFs). (See theExamples section also). The C3 transferase is administered in proteinform, or more preferably, its cDNA is expressed using an expressionvector in the cells of interest of a subject according to the invention.Rho GTPase-specific guanine nucleotide exchange factor inhibitorsinclude chemicals, antisense nucleic acids, antibodies, catalyticnucleic acids including ribozymes, proteins which repress expression ofa rho GTPase-specific guanine nucleotide exchange factor gene locus,proteins, peptides (including dominant-negative peptides andantibodies), and the like.

According to the invention, agents that disrupt actin cytoskeletalorganization are used excluding rho GTPase function inhibitors as agentsuseful in upregulating ecNOS activity. The invention can involve use ofa rho GTPase function inhibitor (including a HMG-CoA reductaseinhibitor), however, only if used together with an agent that disruptsactin cytoskeletal organization other than a rho GTPase functioninhibitor.

HMG-CoA reductase inhibitors inhibit post-translational modifications ofrho GTPases by preventing mevalonate synthesis and consequentlygeranylgeranylpyrophosphate synthesis, an isoprenoid that is attached tothe CAAX motif of rho GTPases. Examples of HMG-CoA reductase inhibitorsinclude some which are commercially available, such as simvastatin (U.S.Pat. No. 4, 444,784), lovastatin (U.S. Pat. No. 4,231,938), pravastatinsodium (U.S. Pat. No. 4,346,227), fluvastatin (U.S. Pat. No. 4,739,073),atorvastatin (U.S. Pat. No. 5,273,995), cerivastatin, and numerousothers described in U.S. Pat. No. 5,622,985, U.S. Pat. No. 5,135,935,U.S. Pat. No. 5,356,896, U.S. Pat. No. 4,920,109, U.S. Pat. No.5,286,895, U.S. Pat. No. 5,262,435, U.S. Pat. No. 5,260,332, U.S. Pat.No. 5,317,031, U.S. Pat. No. 5,283,256, U.S. Pat. No. 5,256,689, U.S.Pat. No. 5,182,298, U.S. Pat. No. 5,369,125, U.S. Pat. No. 5,302,604,U.S. Pat. No. 5,166,171, U.S. Pat. No. 5,202,327, U.S. Pat. No.5,276,021, U.S. Pat. No. 5,196,440, U.S. Pat. No. 5,091,386, U.S. Pat.No. 5,091,378, U.S. Pat. No. 4,904,646, U.S. Pat. No. 5,385,932, U.S.Pat. No. 5,250,435, U.S. Pat. No. 5,132,312, U.S. Pat. No. 5,130,306,U.S. Pat. No. 5,116,870, U.S. Pat. No. 5,112,857, U.S. Pat. No.5,102,911, U.S. Pat. No. 5,098,931, U.S. Pat. No. 5,081,136, U.S. Pat.No. 5,025,000, U.S. Pat. No. 5,021,453, U.S. Pat. No. 5,017,716, U.S.Pat. No. 5,001,144, U.S. Pat. No. 5,001,128, U.S. Pat. No. 4,997,837,U.S. Pat. No. 4,996,234, U.S. Pat. No. 4,994,494, U.S. Pat. No.4,992,429, U.S. Pat. No. 4,970,231, U.S. Pat. No. 4,968,693, U.S. Pat.No. 4,963,538, U.S. Pat. No. 4,957,940, U.S. Pat. No. 4,950,675, U.S.Pat. No. 4,946,864, U.S. Pat. No. 4,946,860, U.S. Pat. No. 4,940,800,U.S. Pat. No. 4,940,727, U.S. Pat. No. 4,939,143, U.S. Pat. No.4,929,620, U.S. Pat. No. 4,923,861, U.S. Pat. No. 4,906,657, U.S. Pat.No. 4,906,624 and U.S. Pat. No. 4,897,402, the disclosures of whichpatents are incorporated herein by reference.

Other rho GTPase function inhibitors not described in the abovecategories and useful according to the invention include agents thatinhibit rho GTPase activation via a receptor-mediated signaling pathway.Such agents include protein kinase C inhibitors, Gq protein inhibitors(e.g., C-terminal antibodies, dominant-negative Gq mutants, etc.),tyrosine kinase inhibitors (e.g., genistein, etc.), tyrosine phosphatasestimulators, GTPase-activating protein stimulators, inhibitors ofintegrins and adhesion molecules, adapter protein (Shc and Sos)inhibitors, and Pleckstrin homology domains which bind G-protein bg.

The invention also involves the co-administration of agents that are notagents that disrupt actin cytoskeletal organization but that can actcooperatively, additively or synergistically with such agents thatdisrupt actin cytoskeletal organization to increase ecNOS activity.Thus, ecNOS substrates which are converted by ecNOS to nitric oxide canbe co-administered with the agents that disrupt actin cytoskeletalorganization according to the invention. Such ecNOS substrates may benatural or synthetic, although the preferred substrate is L-arginine.

Likewise, there are other agents besides agents that disrupt actincytoskeletal organization, that are not substrates of ecNOS, and thatcan increase ecNOS activity. Examples of categories of such agents areestrogens and ACE inhibitors. Estrogens are a well defined category ofmolecules known by those of ordinary skill in the art, and will not beelaborated upon further herein. All share a high degree of structuralsimilarity. ACE inhibitors also have been well characterized, althoughthey do not always share structural homology.

Angiotensin converting enzyme, or ACE, is an enzyme which catalyzes theconversion of angiotensin I to angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di and tripeptides and antibodies to ACE which intervene in the renin-angiotensinsystem by inhibiting the activity of ACE thereby reducing or eliminatingthe formation of pressor substance angiotensin II. ACE inhibitors havebeen used medically to treat hypertension, congestive heart failure,myocardial infarction and renal disease. Classes of compounds known tobe useful as ACE inhibitors include acylmercapto and mercaptoalkanoylprolines such as captopril (U.S. Pat. No. 4,105,776) and zofenopril(U.S. Pat. No. 4,316,906), carboxyalkyl dipeptides such as enalapril(U.S. Pat. No. 4,374,829), lisinopril (U.S. Pat. No. 4,374,829),quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S. Pat. No. 4,587,258),and perindopril (U.S. Pat. No. 4,508,729), carboxyalkyl dipeptide mimicssuch as cilazapril (U.S. Pat. No. 4,512,924) and benazapril (U.S. Pat.No. 4,410,520), phosphinylalkanoyl prolines such as fosinopril (U.S.Pat. No. 4,337,201) and trandolopril.

Estrogens upregulate Nitric Oxide Synthase expression whereas ACEinhibitors do not affect expression, but instead influence theefficiency of the action of Nitric Oxide Synthase on L-arginine. Thus,activity can be increased in a variety of ways. In general, activity isincreased by the reductase inhibitors of the invention by increasing theamount of the active enzyme present in a cell versus the amount presentin a cell absent treatment with the reductase inhibitors according tothe invention.

The invention also involves the co-administration of “at least onedifferent agent that disrupts actin cytoskeletal organization” (secondagent that disrupts actin cytoskeletal organization) that can actcooperatively, additively or synergistically with a first agent thatdisrupts actin cytoskeletal organization of the invention to increaseecNOS activity. Thus, “at least one different agent that disrupts actincytoskeletal organization” is meant to include one or more agent(s) thatdisrupts actin cytoskeletal organization that is (are) different to thefirst agent that disrupts actin cytoskeletal organization of theinvention and can include a HMG-CoA reductase inhibitor and/or a rhoGTPase function inhibitor. In one embodiment, when the agent thatdisrupts actin cytoskeletal organization according to the invention isco-administered in combination with “at least one different agent thatdisrupts actin cytoskeletal organization” and the “at least onedifferent agent that disrupts actin cytoskeletal organization” is aHMG-CoA reductase inhibitor, the subject is nonhypercholesterolemic.

The agents that disrupt actin cytoskeletal organization are administeredin effective amounts. In general, an effective amount is any amount thatcan cause an increase in Nitric Oxide Synthase activity in a desiredcell or tissue, and preferably in an amount sufficient to cause afavorable phenotypic change in a condition such as a lessening,alleviation or elimination of a symptom or of a condition.

In general, an effective amount is that amount of a pharmaceuticalpreparation that alone, or together with further doses orco-administration of other agents, produces the desired response. Thismay involve only slowing the progression of the disease temporarily,although more preferably, it involves halting the progression of thedisease permanently or delaying the onset of or preventing the diseaseor condition from occurring. This can be monitored by routine methods.Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50-500mg/kg will be suitable, preferably orally and in one or severaladministrations per day.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. Lower doses willresult from certain forms of administration, such as intravenousadministration. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels ofcompounds. It is preferred generally that a maximum dose be used, thatis, the highest safe dose according to sound medical judgment. It willbe understood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

The agents that disrupt actin cytoskeletal organization useful accordingto the invention may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of reductase inhibitors, which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono-ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the active compound, increasing convenience to thesubject and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer base systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di-and tri-glycerides; hydrogelrelease systems; sylastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the active compound iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) difusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be desirable. Long-termrelease, are used herein, means that the implant is constructed andarranged to delivery therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well-known to those of ordinary skill in the art andinclude some of the release systems described above.

According to another aspect of the invention, a method for increasingblood flow in a tissue of a subject is provided. The method involvesadministering to a subject in need of such treatment a first agent thatdisrupts actin cytoskeletal organization in an amount effective toincrease endothelial cell Nitric Oxide Synthase activity in the tissueof the subject, provided that the first agent is not a rho GTPasefunction inhibitor or fasudil. Fasudil (a substitutedisoquinolinesulfonyl compound- also known as HA1077), described in U.S.Pat. No. 4,678,783 as a compound with vasodilating properties, was notknown to act as an agent that disrupts actin cytoskeletal organizationresulting in increased ecNOS expression prior to the present invention.

In important embodiments a second agent is co-administered to a subjectwith a condition treatable by the second agent in an amount effective totreat the condition, whereby the delivery of the second agent to atissue of the subject is enhanced as a result of the increased bloodflow from administering the first agent of the invention (an agent thatdisrupts actin cytoskeletal organization). The “second agent” may be anypharmacological compound or diagnostic agent, as desired.

Examples of catagories of pharmaceutical agents include: adrenergicagent; adrenocortical steroid; adrenocortical suppressant; alcoholdeterrent; aldosterone antagonist; amino acid; ammonia detoxicant;anabolic; analeptic; analgesic; androgen; anesthesia, adjunct to;anesthetic; anorectic; antagonist; anterior pituitary suppressant;anthelmintic; anti-acne agent; anti-adrenergic; anti-allergic;anti-amebic; anti-androgen; anti-anemic; anti-anginal; anti-anxiety;anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial;anticholelithic; anticholelithogenic; anticholinergic; anticoagulant;anticoccidal; anticonvulsant; antidepressant; antidiabetic;antidiarrheal; antidiuretic; antidote; anti-emetic; anti-epileptic;anti-estrogen; antifibrinolytic; antifungal; antiglaucoma agent;antihemophilic; antihemorrhagic; antihistamine; antihyperlipidemia;antihyperlipoproteinemic; antihypertensive; anti-infective;anti-infective, topical; anti-inflammatory; antikeratinizing agent;antimalarial; antimicrobial; antimigraine; antimitotic; antimycotic,antinauseant, antineoplastic, antineutropenic, antiobessional agent;antiparasitic; antiparkinsonian; antiperistaltic, antipneumocystic;antiproliferative; antiprostatic hypertrophy; antiprotozoal;antipruritic; antipsychotic; antirheumatic; antischistosomal;antiseborrheic; antisecretory; antispasmodic; antithrombotic;antitussive; anti-ulcerative; anti-urolithic; antiviral; appetitesuppressant; benign prostatic hyperplasia therapy agent; blood glucoseregulator; bone resorption inhibitor; bronchodilator; carbonic anhydraseinhibitor; cardiac depressant; cardioprotectant; cardiotonic;cardiovascular agent; choleretic; cholinergic; cholinergic agonist;cholinesterase deactivator; coccidiostat; cognition adjuvant; cognitionenhancer; depressant; diagnostic aid; diuretic; dopaminergic agent;ectoparasiticide; emetic; enzyme inhibitor; estrogen; fibrinolytic;fluorescent agent; free oxygen radical scavenger; gastrointestinalmotility effector; glucocorticoid; gonad-stimulating principle; hairgrowth stimulant; hemostatic; histamine H2 receptor antagonists;hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive;imaging agent; immunizing agent; immunomodulator; immunoregulator;immunostimulant; immunosuppressant; impotence therapy adjunct;inhibitor; keratolytic; LNRH agonist; liver disorder treatment;luteolysin; memory adjuvant; mental performance enhancer; moodregulator; mucolytic; mucosal protective agent; mydriatic; nasaldecongestant; neuromuscular blocking agent; neuroprotective; NMDAantagonist; non-hormonal sterol derivative; oxytocic; plasminogenactivator; platelet activating factor antagonist; platelet aggregationinhibitor; post-stroke and post-head trauma treatment; potentiator;progestin; prostaglandin; prostate growth inhibitor; prothyrotropin;psychotropic; pulmonary surface; radioactive agent; regulator; relaxant;repartitioning agent; scabicide; sclerosing agent; sedative;sedative-hypnotic; selective adenosine Al antagonist; serotoninantagonist; serotonin inhibitor; serotonin receptor antagonist; steroid;stimulant; suppressant; symptomatic multiple sclerosis; synergist;thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer;treatment of amyotrophic lateral sclerosis; treatment of cerebralischemia; treatment of Paget's disease; treatment of unstable angina;uricosuric; vasoconstrictor; vasodilator; vulnerary; wound healingagent; xanthine oxidase inhibitor.

In another aspect of the invention, the agent that disrupts actincytoskeletal organization is “co-administered,” which means administeredsubstantially simultaneously with another agent. By substantiallysimultaneously, it is meant that the agent that disrupts actincytoskeletal organization is administered to the subject close enough intime with the administration of the other agent, whereby the twocompounds may exert an additive or even synergistic effect, i.e. onincreasing ecNOS activity or on delivering a second agent to a tissuevia increased blood flow.

EXAMPLES

“Upregulation of endothelial cell Nitric Oxide Synthase by HMG CoAReductase Inhibitors”

Experimental Procedures

All standard culture reagents were obtained from JRH Bioscience (Lenexa,Kans.). Unless indicated otherwise, all reagents were purchased fromSigma Chemical Co. (St. Louis, Mo.). [a-³²P]CTP (3000 Ci/mmol) wassupplied by New England Nuclear. Purified human LDL was obtained fromCalbiochem (San Diego, Calif.; lot#730793) and Biomedical TechnologiesInc. (Stoughton, Mass.; lot#9030197). The level of endotoxin wasdetermined by the chromogenic Limulus amebocyte assay (BioWhittakerInc., Walkersville, Md.). The antibody detection kit (EnhancedChemiluminescence) and the nylon nucleic acid (Hybond) and protein(PVDF) transfer membranes were purchased from Amersham Corp. (ArlingtonHeights, Ill.). Simvastatin and lovastatin were obtained from Merck,Sharp, and Dohme, Inc. (West Point, Pa.). Since endothelial cells lacklactonases to process simvastatin and lovastatin to their active forms,these HMG-CoA reductase inhibitors were chemically activated prior totheir use with methods well known in the art and as previously described(Laufs, U et al., J Biol Chem, 1997, 272:31725-31729).

Cell Culture

Human endothelial cells were harvested from saphenous veins and culturedas described (15). For transfection studies, bovine aortic endothelialcells of less than 3 passages were cultured in a growth mediumcontaining DMEM (Dulbecco's Modified Eagle's Medium), 5 mmol/LL-glutamine (Gibco), and 10% fetal calf serum (Hyclone Lot#1114577). Forall experiments, the endothelial cells were placed in 10%lipoprotein-deficient serum (Sigma, Lot#26H94031) for 48 h prior totreatment conditions. In the indicated experiments, endothelial cellswere pretreated with actinomycin D (5 mg/ml) for 1 h prior to treatmentwith ox-LDL and/or simvastatin. Cellular viability as determined by cellcount, morphology, and Trypan blue exclusion was maintained for alltreatment conditions.

Preparation of LDL

The LDL was prepared by discontinuous ultracentrifugation according tothe method of Chung et al. with some modification (Methods Enzymol,1984, 128:181-209). Fresh plasma from a single donor was anticoagulatedwith heparin and filtered through a Sephadex G-25 column equilibratedwith PBS. The density was adjusted to 1.21 g/ml by addition of KBr(0.3265 g/ml plasma). A discontinuous NaCl/KBr gradient was establishedin Beckman Quick-Seal centrifuge tubes (5.0 ml capacity) by layering 1.5ml of density-adjusted plasma under 3.5 ml of 0.154 M NaCl inChelex-100-treated water (BioRad, Hercules, Calif.). Afterultracentrifugation at 443,000× g and 7° C. for 45 min in a Beckman NearVertical Tube 90 rotor (Beckman L8-80M ultracentrifuge), the yellow bandin the upper middle of the tube corresponding to LDL was removed bypuncturing with a needle and withdrawing into a syringe. The KBr wasremoved from the LDL by dialyzing with three changes of sterile PBS, pH7.4, containing 100 μg/ml polymyxin B.

The purity of the LDL samples was confirmed by SDS/polyacrylamide andcellulose acetate gel electrophoresis. Cholesterol and triglyceridecontent were determined as previously described (Liao, J K et al., JBiol Chem, 1995, 270:319-324.). The LDL protein concentration wasdetermined by the method of Lowry et al., (J Biol Chem, 1951,193:265-275.). For comparison, commercially-available LDL (BiomedicalTechnologies Inc., Stoughton, M A; Calbiochem, San Diego, Calif.) werecharacterized and used in selected experiments.

Oxidation of LDL

Oxidized LDL was prepared by exposing freshly-isolated LDL to CuSO₄(5-10 mM) at 37° C. for various duration (6-24 h). The reaction wasstopped by dialyzing with three changes of sterile buffer (150 μmol/LNaCl, 0.01% EDTA and 100 μg/ml polymyxin B, pH 7.4) at 4° C. The degreeof LDL oxidation was estimated by measuring the amounts ofthiobarbituric acid reactive substances (TBARS) produced using afluorescent assay for malondialdehyde as previously described (Yagi, KA, Biochem Med, 1976, 15:212-21.). The extent of LDL modification wasexpressed as nanomoles of malondialdehyde per mg of LDL protein. Onlymild to moderate ox-LDL with TBARS values between 12 and 16 nmol/mg LDLprotein (i.e. 3 to 4 nmol/mg LDL cholesterol) were used in this study.All oxidatively-modified LDL samples were used within 24 h ofpreparation.

Northern Blotting

Equal amounts of total RNA (10-20 mg) were separated by 1.2%formaldehyde-agarose gel electrophoresis and transferred overnight ontoHybond nylon membranes. Radiolabeling of human full-length ecNOS cDNA(Verbeuren, T J et al., Circ Res, 1986, 58:552-564, Liao, J K et al., JClin Invest, 1995, 96:2661-2666) was performed using random hexamerpriming, [a-³²P]CTP, and Klenow (Pharmacia). The membranes werehybridized with the probes overnight at 45° C. in a solution containing50% formamide, 5× SSC, 2.5× Denhardt's Solution, 25 mM sodium phosphatebuffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. All Northernblots were subjected to stringent washing conditions (0.2× SSC/0.1% SDSat 65° C.) prior to autoradiography. RNA loading was determined byrehybridization with human GAPDH probe.

Western Blotting

Cellular proteins were prepared and separated on SDS/PAGE as described(Liao, J K et al., J Biol Chem, 1995, 270:319-324). Immunoblotting wasperformed using a murine monoclonal antibody to human ecNOS (1:400dilution, Transduction Laboratories, Lexington, Ky.). Immunodetectionwas accomplished using a sheep anti-mouse secondary antibody (1:4000dilution) and the enhanced chemiluminescence (ECL) kit (Amersham Corp.,Arlington Heights, Ill.). Autoradiography was performed at 23° C. andthe appropriate exposures were quantitated by densitometry.

Assay for ecNOS Activity

The ecNOS activity was determined by a modified nitrite assay aspreviously described (Misko, T P et al., Analytical Biochemistry, 1993,214:11-16, Liao, J K et al., J Clin Invest, 1995, 96:2661-2666).Briefly, endothelial cells were treated for 24 h with ox-LDL in thepresence and absence of simvastatin (0.1 to 1 mM). After treatment, themedium was removed, and the cells were washed and incubated for 24 h inphenol red-free medium. After 24 h, 300 μl of conditioned medium wasmixed with 30 μl of freshly prepared 2,3-diaminonaphthalene (1.5 mmol/LDAN in 1 mol/L HCl). The mixture was protected from light and incubatedat 20° C. for 10 min. The reaction was terminated with 15 μl of 2.8mol/L NaOH. Fluorescence of 1-(H)-naphthotriazole was measured withexcitation and emission wavelengths of 365 and 450 nm, respectively.Standard curves were constructed with known amounts of sodium nitrite.Nonspecific fluorescence was determined in the presence of LNMA (5mmol/L).

Nuclear Run-on Assay

Confluent endothelial cells (˜5×10⁷ cells) grown in LPDS were treatedwith simvastatin (1 mM) or 95%O₂ for 24 h. Nuclei were isolated and invitro transcription was performed as previously described (Liao, J K etal., J Clin Invest, 1995, 96:2661-2666). Equal amounts (1 mg) ofpurified, denatured full-length human ecNOS, human b-tubulin (ATCC#37855), and linearized pGEM-3z cDNA were vacuum-transferred ontonitrocellulose membranes using a slot blot apparatus (Schleicher &Schuell). Hybridization of radiolabeled mRNA transcripts to thenitrocellulose membranes was carried out at 45° C. for 48 h in a buffercontaining 50% formamide, 5× SSC, 2.5× Denhardt's solution, 25 mM sodiumphosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. Themembranes were then washed with 1× SSC/0.1% SDS for 1 h at 65° C. priorto autoradiography for 72 h at −80° C.

Transfection Assays

For transient transfections, bovine rather than human endothelial cellswere used because of their higher transfection efficiency by thecalcium-phosphate precipitation method (12% vs<4%) (Graham, F L and Vander Erb, A J, Virology, 1973, 52:456-457). We used the human ecNOSpromoter construct, F1.LUC, which contains a −1.6 kb 5′-upstreamsequence linked to the luciferase reporter gene as described by Zhang etal. (J Biol Chem, 1995, 270:15320-15326). Bovine endothelial cells(60%-70% confluent) were transfected with 30 mg of the indicatedconstructs: p.LUC (no promoter), pSV2.LUC (SV40 early promoter), orF1.LUC. As an internal control for transfection efficiency, pCMV.bGalplasmid (10 mg) was co-transfected in all experiments. Preliminaryresults using b-galactosidase staining indicate that cellulartransfection efficiency was approximately 10% to 14%.

Endothelial cells were placed in lipoprotein-deficient serum for 48 hafter transfection and treated with ox-LDL (50 mg/ml, TBARS 12.4nmol/mg) in the presence and absence of simvastatin (1 mM) for anadditional 24 h. The luciferase and b-galactosidase activities weredetermined by a chemiluminescence assay (Dual-Light, Tropix, Bedford,Mass.) using a Berthold L9501 luminometer. The relative promoteractivity was calculated as the ratio of luciferase- to b-galactosidaseactivity. Each experiment was performed three times in triplicate.

Data Analysis

Band intensities were analyzed densitometrically by the NationalInstitutes of Health Image program (Rasband, W, NIH Image program, v1.49, National Institutes of Health, Bethesda, 1993). All values areexpressed as mean ± SEM compared to controls and among separateexperiments. Paired and unpaired Student's t tests were employed todetermine any significant changes in densitometric values, nitriteproduction, and promoter activities. A significant difference was takenfor P values less than 0.05.

EXAMPLE 1 Cell Culture

Relatively pure (>95%) human endothelial cell cultures were confirmed bytheir morphological features (i.e. cuboidal, cobble-stone, contactinhibited) using phase-contrast microscopy and by immunofluorescentstaining with anti-Factor VIII antibodies (Gerson, R J et al., Am J Med,1989, 87:28-38). For all experimental conditions, there were noobservable adverse effects of ox-LDL or HMG-CoA reductase inhibitors oncellular morphology, cell number, immunofluorescent staining, and Trypanblue exclusion (>95%). Higher concentrations of ox-LDL (>100 mg/ml) withgreater oxidative modification (i.e. TBARS values of >30 nmol/mg) causedvacuolization and some detachment of endothelial cells after 24 h.Neither simvastatin (0.01 to 0.1 mmol/L) nor lovastatin (10 mmol/L)produced any noticeable adverse effects on human endothelial cell for upto 96 h. However, higher concentrations of simvastatin (>15 mmol/L) orlovastatin (>50 mmol/L) caused cytotoxicity after 36 h, and therefore,were not used.

EXAMPLE 2 Characterization of LDL

SDS/polyacrylamide gel electrophoresis of native or unmodified LDLrevealed a single band (˜510 kD) corresponding to ApoB-100 (data notshown). Similarly, cellulose acetate electrophoresis revealed only oneband corresponding to the presence of a single class of low-densitylipids (density of 1.02 to 1.06 g/ml). The LDL had a protein,cholesterol, and triglyceride concentration of 6.3±0.2, 2.5±0.1, and0.5±0.1 mg/ml, respectively. In contrast, lipoprotein-deficient serumwas devoid of both apoB-100 protein and low-density lipid bands, and hadnon-detectable levels of cholesterol. There was no detectable level ofendotoxin (<0.10 EU/ml) in the lipoprotein-deficient serum or ox-LDLsamples by the chromogenic Limulus amebocyte assay.

In addition, there was no apparent difference between our ownpreparation and commercially-obtained LDL samples in terms ofelectrophoretic mobility. Native LDL had a TBARS value of 0.3±0.2nmol/mg, but after exposure to human saphenous vein endothelial cells inlipoprotein-deficient media for 72 h, this value increased to 3.1±0.4nmol/mg. Copper-oxidized LDL had TBARS values ranging from 4.6±0.5 to33.1±5.2 nmol/mg. The degree of ox-LDL used in this study was mild tomoderate with TBARS value ranging from 12 to 16 nmol/mg LDL protein(i.e. 3 to 4 nmol/mg LDL cholesterol).

EXAMPLE 3 Effect of ox-LDL and HMG-CoA Reductase Inhibitors on ecNOSProtein

We have previously shown that ox-LDL (50 mg/ml) downregulates ecNOSexpression (Liao, J K et al., J Biol Chem, 1995, 270:319-324). Comparedto untreated cells, treatment with ox-LDL (50 mg/ml, TBARS 12.2 nmol/mg)caused a 54%±6% decrease in ecNOS protein after 48 h (p<0.01, n=4).There was no difference between our preparation of ox-LDL andcommercially-available ox-LDL with similar TBARS values in terms of thedegree of ecNOS downregulation. Addition of simvastatin (0.01 mmol/L)did not significantly affect the downregulation of ecNOS protein byox-LDL (57%±8% decrease, p>0.05, n=4). However, in the presence of 0.1mmol/L of simvastatin, ox-LDL no longer produce any significant decreasein ecNOS protein levels (4%±7% decrease, p<0.01, n=4). Higherconcentrations of simvastatin (1 and 10 mmol/L) resulted in not only areversal of ecNOS downregulation by ox-LDL, but also significantincreases in ecNOS protein levels above baseline (146%±9% and 210% 112%,respectively, p<0.05, n=4). Simvastatin or lovastatin (10 mmol/L) whichwere not chemically-activated had no effect on ecNOS expression.

In a time-dependent manner, treatment with ox-LDL (50 mg/ml, TBARS 12.2nmol/mg) decreased ecNOS protein expression by 34% (5%, 67% (8% and 86(5% after 24 h, 72 h, and 96 h, respectively (p<0.05 for all values,n=4,). Compared to ox-LDL alone, co-treatment with simvastatin (0.1mmol/L) attenuated the decrease in ecNOS protein level after 24 h (15%(2% vs 34% (5%, p<0.05, n=4). Longer incubation with simvastatin (0.1mmol/L) for 72 h and 96 h not only reversed ox-LDL's inhibitory effectson ecNOS expression, but also increased ecNOS protein levels by 110% (6%and 124% (6% above basal expression (p<0.05, n=4). Thus, compared toox-LDL alone, co-treatment with simvastatin produced a 1.3-, 3.3-and8.9-fold increase ecNOS protein levels after 24 h, 72 h, and 96 h,respectively.

EXAMPLE 4 Effect of ox-LDL and HMG-CoA Reductase Inhibitors on ecNOSmRNA

The effect of simvastatin on ecNOS mRNA levels occurred in atime-dependent manner and correlated with its effect on ecNOS proteinlevels. Northern analyses showed that ox-LDL (50 mg/ml, TBARS 15.1nmol/mg) produced a time-dependent 65±5% and 91±4% decrease in ecNOSmRNA levels after 48 h and 72 h, respectively (p<0.01, n=3). Compared toox-LDL at the indicated time points, co-treatment with simvastatin 0.1mmol/L) increased ecNOS mRNA levels by 6.3-fold after 48 h and 14.5-foldafter 72 h (p<0.01 for all values, n=3).

To determine whether treatment with another HMG-CoA reductase inhibitorhave similar effect as simvastatin, we treated endothelial cells withlovastatin. Again, ox-LDL decreased steady-state ecNOS mRNA by 52±5%after 24 h (p<0.01, n=3). Treatment with lovastatin (10 mmol/L) not onlyreversed the inhibitory effects of ox-LDL on ecNOS mRNA, but also causeda 40±9% increase in ecNOS mRNA level compared to that of untreatedcells. Compared to ox-LDL alone, co-treatment with lovastatin caused a3.6-fold increase in ecNOS mRNA levels after 24 h. Treatment withlovastatin alone, however, produced 36% increase in ecNOS mRNA levelscompared to untreated cells (p<0.05, n=3).

EXAMPLE 5 Effect of ox-LDL and Simvastatin on ecNOS Activity

The activity of ecNOS was assessed by measuring the LNMA-inhibitablenitrite production from human endothelial cells (Liao, J K et al., JClin Invest, 1995, 96:2661-2666). Basal ecNOS activity was 8.8±1.4nmol/500,000 cells/24 h. Treatment with ox-LDL (50 mg/ml, TBARS 16nmol/mg) for 48 h decreased ecNOS-dependent nitrite production by 94±3%(0.6±0.5 nmol/500,000 cells/24 h, p<0.001). Co-treatment withsimvastatin (0.1 mmol/L) significantly attenuated this downregulationresulting in a 28±3% decrease in ecNOS activity compared to untreatedcells (6.4±0.3 nmol/500,000 cells/24 h, p<0.05). Co-treatment with ahigher concentration of simvastatin (1 mmol/L) not only completelyreversed the downregulation of ecNOS by ox-LDL, but also, resulted in a45±6% increase in ecNOS activity compared to baseline (12.8±2.7nmol/500,000 cells/24 h, p<0.05).

EXAMPLE 6 Effect of Simvastatin on ecNOS mRNA Stability

The post-transcriptional regulation of ecNOS mRNA was determined in thepresence of the transcriptional inhibitor, actinomycin D (5 mg/ml).Oxidized LDL (50 mg/ml, TBARS 13.1 nmol/mg) shortened the half-life ofecNOS mRNA (t1/2 35±3 h to 14±2 h, p<0.05, n=3). Co-treatment withsimvastatin (0.1 mmol/L) prolonged the half-life of ecNOS mRNA by1.6-fold (t1/2 22±3 h, p<0.05, n=3). Treatment with simvastatin aloneprolonged ecNOS mRNA half-life by 1.3-fold over baseline (t1/2 43±4 h,p<0.05, n=3).

EXAMPLE 7 Effect of Simvastatin on ecNOS Gene Transcription

To determine whether the effects of simvastatin on ecNOS expressionoccurs at the level of ecNOS gene transcription, we performed nuclearrun-on assays using endothelial cells treated with simvastatin (1mmol/L) for 24 h. Preliminary studies using different amounts ofradiolabelled RNA transcripts demonstrate that under our experimentalconditions, hybridization was linear and nonsaturable. The density ofeach ecNOS band was standardized to the density of its correspondingb-tubulin. The specificity of each band was determined by the lack ofhybridization to the nonspecific pGEM cDNA vector. In untreatedendothelial cells (control), there was constitutive ecNOStranscriptional activity (relative index of 1.0). Treatment withsimvastatin (1 mmol/L) did not significantly affect ecNOS genetranscription compared to that of untreated cells (relative index of1.2±0.3, p>0.05, n=4). However, treatment of endothelial cells withhyperoxia (95%_(O2)) significantly increased ecNOS gene expression(relative index of 2.5, p<0.05, n=4).

To further confirm the effects of simvastatin on ecNOS genetranscription by a different method, we transfected bovine aorticendothelial cells using a −1600 to +22 nucleotide ecNOS 5′-promoterconstruct linked to a luciferase reporter gene (F 1.LUC) (Zhang, R etal., J Biol Chem, 1995, 270:15320-15326). This promoter constructcontains putative cis-acting elements for activator protein (AP)-1 and-2, sterol regulatory element-1, retinoblastoma control element, shearstress response element (SSRE), nuclear factor-1 (NF-1), and cAMPresponse element (CRE). Treatment with ox-LDL (50 mg/ml, TBARS 14.5nmol/mg), simvastatin (1 μmol/L), alone or in combination, did notsignificantly affect basal F1 promoter activity. However, laminar fluidshear-stress (12 dynes/cm2 for 24 h) was able to induce F1 promoteractivity by 16-fold after 24 h (data not shown) indicating that the F1promoter construct is functionally-responsive if presented with theappropriate stimulus.

EXAMPLE 8 Effect of Simvastatin and Lovastatin on ecNOS Expression

To further characterize the effects of HMG-CoA reductase inhibitors onthe upregulation ecNOS expression, we treated endothelial cells withsimvastatin (0.1 mmol/L) for various durations (0-84 h). Treatment withsimvastatin (0.1 mmol/L) increased ecNOS protein levels by 4 (6%, 21(9%, 80 (8%, 90 (12%, and 95 (16% after 12 h, 24 h, 48 h, 72 h, and 84h, respectively (p<0.05 for all time points after 12 h, n=4). Higherconcentrations of simvastatin similarly increased ecNOS protein levels,but in significantly less time compared to lower concentrations ofsimvastatin (data not shown).

In a concentration-dependent manner, treatment with simvastatin (0.01 to10 mmol/L, 48 h) increased ecNOS expression by 1 (6%, 80 (8%, 190 (10%and 310 (20%, respectively (p<0.05 for concentrations (0.1 mmol/L, n=4).The upregulation of ecNOS expression by simvastatin, therefore, isdependent upon both the concentration and duration of simvastatintreatment. For comparison, treatment with lovastatin (0.1 to 10 mmol/L,48 h) also increased ecNOS expression in a concentration-dependantmanner (10 (6%, 105 (8% and 180 (11%, respectively, p<0.05 forconcentrations>0.1 mmol/L, n=3) but significantly less effectively thansimvastatin at comparable concentrations. Therefore, at the sameconcentration, simvastatin had greater effects on ecNOS expressioncompared to lovastatin. These results are consistent with reported IC50values for simvastatin and lovastatin (4 nmol/L and 19 nmol/L,respectively) (Van Vliet, A K et al., Biochem Pharmacol, 1996,52:1387-1392).

EXAMPLE 9 Effect of L-Mevalonate on ecNOS Expression

To confirm that the effects of simvastatin on ecNOS expression were dueto the inhibition of endothelial HMG CoA reductase, endothelial cellswere treated with ox-LDL (50 mg/ml, TBARS 15.1 nmol/mg), simvastatin (1mmol/L), alone or in combination, in the presence of L-mevalonate (100mmol/L). Treatment with ox-LDL decreased ecNOS expression by 55% (6%after 48 h which was completely reversed and slightly upregulated in thepresence of simvastatin (1 mmol/L) (150% (8% above basal expression)(p<0.05 for both, n=3).

Compared to endothelial cells treated with ox-LDL and simvastatin,addition of L-mevalonate reduced ecNOS protein by 50%±5% (p<0.05, n=3).Furthermore, the upregulation of ecNOS expression by simvastatin alone(2.9-fold increase, p<0.05, n=3) was completely reversed by co-treatmentwith L-mevalonate. Treatment with L-mevalonate alone did not have anyappreciable effects on basal ecNOS expression (p>0.05, n=3). Similarfindings were also observed with L-mevalonate and lovastatin.

“HMG-CoA Reductase Inhibitors Reduce Cerebral Infarct Size byUpregulating endothelial cell Nitric Oxide Synthase”

Experimental Procedures

Cell Culture

Human endothelial cells were harvested from saphenous veins using TypeII collagenase (Worthington Biochemical Corp., Freehold, N.J.) aspreviously described. Cells of less than three passages were grown toconfluence in a culture medium containing Medium 199, 20 mM HEPES, 50mg/ml ECGS (Collaborative Research Inc., Bedford, Mass.), 100 mg/mlheparin sulfate, 5 mM L-glutamine (Gibco), 5% fetal calf serum (Hyclone,Logan, Utah.), and antibiotic mixture of penicillin (100U/ml)/streptomycin (100 mg/ml)/Fungizone (1.25 mg/ml). For allexperiments, the endothelial cells were grown to confluence before anytreatment conditions. In some experiments, cells were pretreated withactinomycin D (5 mg/ml) for 1 h prior to treatment with HMG-CoAreductase inhibitors.

Exposure of Endothelial Cells to Hypoxia

Confluent endothelial cells grown in 100 mm culture dishes were treatedwith HMG-CoA reductase inhibitors and then placed without culture dishcovers in humidified airtight incubation chambers (Billups-Rothenberg,Del Mar, Calif.). The chambers were gassed with 20% or 3% O₂, 5% CO₂,and balanced nitrogen for 10 min prior to sealing the chambers. Thechambers were maintained in a 37° C. incubator for various durations(0-48 h) and found to have less than 2% variation in O₂ concentration aspreviously described (Liao, J K et al., J Clin Invest, 1995,96:2661-2666). Cellular confluence and viability were determined by cellcount, morphology, and trypan blue exclusion.

In vitro Transcription Assay

Confluent endothelial cells (5×10⁷ cells were treated with simvastatin(1 mM) in the presence of 20% or 3% O₂ for 24 h. Nuclei were isolatedand in vitro transcription was performed as previously described (Liao,J K et al., J Clin Invest, 1995, 96:2661-2666). Equal amounts (1 mg) ofpurified, denatured full-length human ecNOS, human b-tubulin (ATCC#37855), and linearized pGEM-3z cDNA were vacuum-transferred ontonitrocellulose membranes using a slot blot apparatus (Schleicher &Schuell). Hybridization of radiolabeled mRNA transcripts to thenitrocellulose membranes was carried out at 45° C. for 48 h in a buffercontaining 50% formamide, 5× SSC, 2.5× Denhardt's solution, 25 mM sodiumphosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. Themembranes were then washed with 1× SSC/0.1% SDS for 1 h at 65° C. priorto autoradiography for 72 h at −80° C. Band intensities were subjectedto analyses by laser densitometry.

Assay for Nitrite Accumulation

The amount of NO produced by ecNOS was determined by nitriteaccumulation in the conditioned medium. Nitrite accumulation wasdetermined by measuring the conversion of 2,3-diaminonaphthalene (1.5 mMof DAN in 1 M of HCl) and nitrite to 1-(H)-naphthotriazole as previouslydescribed (13, 24). Nonspecific fluorescence was determined in thepresence of LNMA (5 mM). Previous studies with nitrate reductaseindicate that the nitrite to nitrate concentration in the medium wasapproximately 5:1 and that this ratio did not vary with exposure to 20%or 3% O₂ concentration.

Murine Model of Cerebral Vascular Ischemia

Adult male (18-20 g) wildtype SV-129 mice (Taconic farm, Germantown,N.Y.) and ecNOS mutant mice (Huang, P L et al., Nature, 1995,377:239-242.) were subcutaneously-injected with 0.2, 2, or 20 mg ofactivated simvastatin per kg body weight or saline (control) once dailyfor 14 days. Ischemia was produced by occluding the left middle cerebralartery (MCA) with a coated 8.0 nylon monofilament under anesthesia asdescribed (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987,Huang, Z et al., Science, 1994, 265:1883-1885, Hara, H et al., J CerebBlood Flow Metab, 1997, 1:515-526). Arterial blood pressure, heart rate,arterial oxygen pressure, and partial pressure of carbon dioxide weremonitored as described (Huang, Z et al., J Cereb Blood Flow Metab, 1996,16:981-987, Huang, Z et al., Science, 1994, 265:1883-1885, Hara, H etal., J Cereb Blood Flow Metab, 1997, 1:515-526). The filaments werewithdrawn after 2 hours and after 24 h, mice were either sacrificed ortested for neurological deficits using a well-established, standardized,observer-blinded protocol as described (Huang, Z et al., J Cereb BloodFlow Metab, 1996, 16:981-987, Huang, Z et al., Science, 1994,265:1883-1885, Hara, H et al., J Cereb Blood Flow Metab, 1997,1:515-526). The motor deficit score range from 0 (no deficit) to 2(complete deficit).

Brains were divided into five coronal 2-mm sections using a mouse brainmatrix (RBM-200C, Activated Systems, Ann Arbor, Mich., USA). Infarctionvolume was quantitated with an image analysis system (M4, St.Catharines, Ontario, Canada) on 2% 2,3,5-triphenyltetrazolium chloridestained 2-mm slices. The levels of serum cholesterol, creatinine andtransaminases were determined by the Tufts University VeterinaryDiagnostic Laboratory (Grafton, Mass.).

Assay for ecNOS Activity from Tissues

The ecNOS activities in mice aortae and brains were measured by theconversion of [³H]arginine to [³H]citrulline in the presence and absenceof LNMA (5 mM) as described earlier.

Quantitative Reverse Transcription-Polymerase Chain Reaction

Total RNA from mouse aortae and brains was isolated by the guanidiniumisothiocyanate method and reverse transcribed using oligo-dT (mRNAPreamplification reagents; Gibco BRL) and Taq ploymerase (Perkin-Elmer).One tenth of the sDNA was used as template for the PCR reaction.Approximately 0.2 nmol of the following primers amplifying a 254-bpfragment of murine ecNOS cDNA were used: 5′Primer:5′-GGGCTCCCTCCTTCCGGCTGCCACC-3′ (SEQ ID NO. 1) and 3′Primer:5′-GGATCCCTGGAAAAGGCGGTGAGG-3′ (SEQ ID NO. 2) (Hara, H et al., J CerebBlood Flow Metab, 1997, 1:515-526). For amplification of glyceraldehyde3-phosphate dehydrogenase (GAPDH), 0.1 nmol of the following primersamplifying a 452-bp fragment were used: 5′Primer:5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO. 3) and 3′ Primer:5′-TCCACCACCCTGTTGCTGTA-3′(SEQ ID NO. 4). Denaturing was performed at94° C. for 30 s, annealing at 60° C. for 30 s, and elongation at 72° C.for 60 s. Preliminary results indicated that the linear exponentialphase for ecNOS and GAPDH polymerization was 30-35 cycles and 20-25cycles, respectively.

EXAMPLE 10 Cell Culture

Relatively pure (>98%) human saphenous vein endothelial cell cultureswere confirmed by their morphological features (ie. cuboidal,cobble-stone, contact inhibited) using phase-contrast microscopy andimmunofluorescent-staining with antibodies to Factor VIII. There were noobservable adverse effects of HMG-CoA reductase inhibitors, L-mevalonicacid, or hypoxia on cellular morphology. However, higher concentrationsof simvastatin (>15 mmol/L) or lovastatin (>50 mmol/L) causedcytotoxicity after 36 h, and therefore, were not used. Otherwise,cellular confluency and viability as determined by trypan blue exclusionwere maintained for all treatment conditions described.

EXAMPLE 11 Effects of HMG-CoA Reductase Inhibitors on ecNOS Activity

The activity of ecNOS was assessed by measuring the LNMA-inhibitablenitrite accumulation from human endothelial cells (Liao, J K et al., JClin Invest, 1995, 96:2661-2666). The ratio of nitrite to nitrateproduction under our culture condition was approximately 5:1 and wassimilar for hypoxia and normoxia (data not shown). Basal ecNOS activityat 20% O₂ was 6.0±3.3 nmol/500,000 cells/24 h. Exposure of endothelialcells to 3% O₂ for 24 h decreased nitrite production by 75±14% (1.5±0.9nmol/500,000 cells/24 h, p<0.01). Treatment with simvastatin (1 mM) notonly completely reversed the downregulation of ecNOS by hypoxia, butresulted in a 3-fold increase in ecNOS activity over basal activity(18±5.0 nmol/500,000 cells/24 h, p<0.05). This upregulation of ecNOSactivity was attenuated by the addition of L-mevalonate (400 mM)(9.6±1.3 nmol/500,000 cells/24 h, p<0.05). Interestingly, simvastatin (1mM) alone upregulated nitrite production 5-fold (30±6.5 nmol/500,000cells/24 h, p<0.01), which was completely blocked by L-mevalonate (400mM) (8.6±2.9 nmol/500,000 cells/24 h, p<0.05). Similar findings wereobserved with lovastatin, but at 10-fold higher concentration comparedto that of simvastatin.

EXAMPLE 12 Effects of HMG-CoA Reductase Inhibitors on ecNOS Protein andmRNA Levels

In a concentration-dependent manner, treatment with simvastatin (0.01 to10 mM, 48 h) increased ecNOS expression by 1 (6%, 80 (8%, 190 (10% and310 (20%, respectively (p <0.05 for concentrations (0.1 mM, n=4).Treatment with simvastatin (0.1 mM) increased ecNOS protein levels in atime-dependent manner by 4 (6%, 21 (9%, 80 (8%, 90 (12%, and 95 (16%after 12 h, 24 h, 48 h, 72 h, and 84 h, respectively (p<0.05 for alltime points after 12 h, n=4) (data not shown). Another HMG-CoA reductaseinhibitor, lovastatin, also increased ecNOS protein levels in a time-,and concentration-dependent manner (data not shown). Because lovastatinhas a higher IC50 value for HMG-CoA reductase compared to that ofsimvastatin, it was 10-fold less potent in upregulating ecNOS proteinlevels than simvastatin at equimolar concentrations.

We have previously shown that hypoxia downregulates ecNOS proteinexpression (Liao, J K et al., J Clin Invest, 1995, 96:2661-2666).Compared to normoxia (20% O₂), exposure to hypoxia (3% O₂) resulted in a46±4% and 75±3% reduction in ecNOS protein levels after 24 h and 48 h,respectively (p<0.0 1, n=3). In a concentration-dependent manner,treatment with simvastatin produced a progressive reversal ofhypoxia-mediated downregulation of ecNOS protein levels after 48 h. Athigher concentrations of simvastatin (1 and 10 mM), ecNOS protein levelswere upregulated to 159±13% and 223±21% of basal levels (p<0.05, n=3).Co-treatment with L-mevalonic acid (400 mM) completely blockedsimvastatin-induced increase in ecNOS protein levels after 48 h(35±2.4%). Treatment with L-mevalonic acid alone, however, did notproduce any significant effects on basal ecNOS protein levels inuntreated cells exposed to hypoxia (25±3.9%, p>0.05, n=3). In addition,simvastatin which was not chemically-activated had no effect on ecNOSexpression. These results indicate that simvastatin- andlovastatin-mediated increases in ecNOS protein expression are mediatedby inhibition of endothelial HMG-CoA reductase.

To determine whether changes in ecNOS protein levels are due to changesin ecNOS steady-state mRNA levels, we performed Northern blotting onendothelial cells exposed to normoxia and hypoxia in the presence orabsence of simvastatin (1 mM) and lovastatin (10 μM). Simvastatin aloneincreased ecNOS mRNA levels to 340±24% (p<0.01, n=3). Exposure ofendothelial cells to hypoxia reduced ecNOS mRNA levels by 70%±2% and88±4% after 24 h and 48 h with respect to GAPDH mRNA levels,respectively. Co-treatment with simvastatin not only completely reversedhypoxia-mediated decrease in ecNOS mRNA levels, but increased ecNOS mRNAlevels to 195±12% and 530±30% of basal levels after 24 h and 48 h,respectively (p<0.01, n=3). Similarly, lovastatin (10 μM) aloneincreased ecNOS message to 350±27% under hypoxia and 410±21% alone(p<0.01, n=3). Neither simvastatin nor lovastatin caused any significantchange in G-protein as and b-actin mRNA levels under normoxic or hypoxicconditions. These results indicate that the effects of HMG-CoA reductaseinhibitors are relatively selective in terms of their effects on ecNOSmRNA expression.

EXAMPLE 13 Effects of HMG-CoA Reductase Inhibitors on ecNOS mRNAHalf-life

The half-life of ecNOS mRNA was determined in the presence ofactinomycin D (5 mg/ml). Hypoxia shortened the half-life of ecNOS mRNAfrom 28±4 h to 13±3 h. Treatment with simvastatin (1 mM) increased ecNOShalf-life to 46±4 h and 38±4 h under normoxic and hypoxic conditions,respectively (p<0.05 for both, n=3). These results suggest that HMG-CoAreductase inhibitors prevent hypoxia-mediated decrease in ecNOSexpression by stabilizing ecNOS mRNA.

EXAMPLE 14 Effects of HMG-CoA Reductase Inhibitors on ecNOS GeneTranscription

Nuclear run-on assays showed that hypoxia caused a 85±8% decrease inecNOS gene transcription (p<0.01, n=3). Treatment with simvastatin (1mM) did not produce any significant affect on hypoxia-mediated decreasein ecNOS gene transcription (83±6% decrease in ecNOS gene transcription,p>0.05 compared to hypoxia alone). Furthermore, simvastatin aloneproduced minimal increase in ecNOS gene transcription under normoxiccondition (20±5% increase in ecNOS gene transcription, p<0.05 comparedto normoxia control).

Preliminary studies using different amounts of radiolabeled RNAtranscripts demonstrate that under our experimental conditions,hybridization was linear and nonsaturable. The density of each ecNOSband was standardized to the density of its corresponding (b-tubulinband, relative intensity). To exclude the possibility that changes in(b-tubulin gene transcription are caused by hypoxia or simvastatin,another gene, GAPDH, was included on each of the nuclear run-on blots.Similar relative indices were obtained when ecNOS gene transcription wasstandardized to GAPDH gene transcription. The specificity of each bandwas determined by the lack of hybridization to the nonspecific pGEM cDNAvector.

EXAMPLE 15 Effect of HMG-CoA Reductase Inhibitors on Mouse Physiology

To determine whether the upregulation of ecNOS by HMG-CoA reductaseinhibitors occurs in vivo, SV-129 wild-type and ecNOS knockout mice weretreated with 2 mg/kg simvastatin or saline subcutaneously for 14 days(n=8). The mean arterial blood pressures of wild-type and ecNOS mutantmice were as reported previously (Huang, PL et al., Nature, 1995,377:239-242). The ecNOS mutants were relatively hypertensive. There wasno significant change in mean arterial blood pressures of wild-type miceafter 14 days of simvastatin treatment (81±7 mmHg vs. 93±10 mmHg,p>0.05). There was also no significant group difference in heart rate,arterial blood gases and temporalis muscle temperature before ischemiaor after reperfusion. Furthermore, there was no significant differencein the levels of serum cholesterol (control: 147±10 vs. simvastatin161±5.2 mg/dl), creatinine and transaminases after treatment withsimvastatin compared to control values.

EXAMPLE 16 Effect of HMG-CoA Reductase Inhibitors on ecNOS Expressionand Function in Mouse Aorta

The activity of ecNOS in the aortae of simvastatin-treated (2 mg/kg) andsaline-injected mice was determined by measuring the LNMA-inhibitableconversion of arginine to citrulline (FIG. 1A). The ecNOS activity inaortae from simvastatin-treated mice was significantly higher than inthe control group (0.39±0.09 vs. 0.18±0.04 U/mg protein, n=8, p<0.05).

The ecNOS mRNA expression in the aortae of simvastatin-treated and-untreated mice was examined by quantitative RT-PCR (FIG. 1B). There wasa significantly dose-dependent 3-fold increase of ecNOS message comparedto that of GAPDH in simvastatin-treated mice (n=3). These findingsindicate that simvastatin upregulates ecNOS expression and activity invivo.

EXAMPLE 17 Effect of HMG-CoA Reductase Inhibitors on Cerebral Ischemiain Mice

Endothelium-derived NO protects against ischemic cerebral injury (Huang,Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987). Therefore weexamined, wether the observed upregulation of ecNOS by simvastatin invivo has beneficial effects on cerebral infarct size. Followingtreatment for 14 days with 2 mg/kg of simvastatin, cerebral ischemia wasproduced by occluding the left middle cerebral artery for 2 hours. After22 hours of reperfusion, mice were tested for neurological deficitsusing a well-established, standardized, observer-blinded protocol. Theneurological motor deficit score improved in simvastatin-treated mice(n=18) by almost 2-fold compared to that of controls (n=12) (0.8±0.2 vs.1.7±0.2, p<0.01).

Simvastatin-treated wild-type mice (n=18) had 25% smaller cerebralinfarct sizes compared to untreated animals (73.8±8.5 mm3 vs. 100.7±7.3mm3, n=12, p<0.05). This effect was concentration-dependent (0.2, 2, 20mg/kg simvastatin), persisted for up to 3 days, and also occurred withlovastatin treatment, albeit at higher relative concentrations (data notshown). Furthermore, simvastatin increase cerebral blood flow by 23% and35% over basal values at concentrations of 2 mg/kg and 20 mg/kg,respectively (n=8, p<0.05 for both). These findings suggest, thatsimvastatin decreases cerebral infarct size and neurological deficits.

Finally, to demonstrate that the reduction of cerebral infarct sizes bysimvastatin is due to the upregulation of ecNOS, cerebral ischemia wasapplied to ecNOS mutant mice lacking ecNOS gene in the presence andabsence of simvastatin (2 mg/kg, 14 days). There was no significantdifference between the cerebral infarct sizes of simvastatin-treated and-untreated ecNOS mutant mice (n=6, p<0.05). These findings indicate thatthe upregulation of ecNOS mediates the beneficial effects of HMG-CoAreductase inhibitors on cerebral infarct size.

EXAMPLE 18 Effect of HMG-CoA Reductase Inhibitors on ecNOS Expression inMouse Brain

The ecNOS mRNA expression in the ischemic and contralateral(non-ischemic) hemispheres of mouse brain was examined by quantitativeRT-PCR (FIG. 2) with respect to GAPDH mRNA levels. Simvastatin-treatedmice (n=3) (2 mg/kg, 14 days) showed a 1.5- to 2-fold increase in ecNOSexpression in the infarcted, ipsilateral hemisphere compared to thecontralateral, non-infarcted side. In contrast, there was no differencein ecNOS expression in untreated mice between their infarcted andnon-infacted hemispheres. These findings suggest that simvastatin mayreduced cerebral infarct size by selectively increasing ecNOS expressionin the ischemic and hypoxic infarct zone.

“Regulation of Endothelial Nitric Oxide Synthase Expression by RhoGTPases”

Experimental Procedures

Materials

Mevastatin, farnesylpyrophosphate, geranylgeranylpyrophosphate, andL-mevalonate were purchased from Sigma Chemical Corp. (St. Louis, Mo.).Mevastatin (compactin-a HMG-CoA reductase inhibitor) was chemicallyactivated by alkaline hydrolysis prior to use as previously described(Laufs, U et al., J Biol Chem, 1997, 272:31725-31729). FPT inhibitor Iand -hydroxyfarnesylphosphonic acid were purchased from Calbiochem Corp.(La Jolla, Calif.). [a-³²P]CTP (3000 Ci/mmol) and [³⁵ S]GTP S (1250Ci/mmol) were supplied by New England Nuclear. The antibody detectionkit (Enhanced Chemiluminescence) and the nylon nucleic acid (Hybond) andprotein (PVDF) transfer membranes were purchased from Amersham Corp.(Arlington Heights, Ill.). The Clostridium botulinum C3 transferase waspurchased from List Biological Laboratories, Inc. (Campbell, Calif.).Recombinant Escherichia coli cytotoxic necrotizing factor (CNF)-1 andRhoA mutants were kindly provided by K. Aktories (University ofFreiberg, Germany) and W. Moolenaar (Netherlands Cancer Institute,Netherlands), respectively.

Cell Culture

Human endothelial cells were harvested using Type II collagenase(Worthington Biochemical Corp., Freehold, N.J.) as previously described(Laufs, U et al., J Biol Chem, 1997, 272:31725-31729; Liao, J K et al.,J Biol Chem, 1995, 270:319-324). Cells of less than three passages weregrown in a culture medium containing Medium 199, 20 mM HEPES, 50 mg/mlECGS (Collaborative Research Inc., Bedford, Mass.), 100 mg/ml heparinsulfate, 5 mM L-glutamine (Gibco), 5% fetal calf serum (Hyclone, Logan,Utah), and antibiotic mixture of penicillin (100 U/ml)/streptomycin (100mg/ml)/Fungizone (1.25 mg/ml). Confluent endothelial cells were used forall treatment conditions. For transfection studies, bovine aorticendothelial cells of less than 3 passages were cultured in a growthmedium containing DMEM (Dulbecco's Modified Eagle's Medium), 5 mM ofL-glutamine (Gibco), and 10% fetal calf serum. Cellular viability wasdetermined by cell count, morphology, and trypan blue exclusion.

Preparation of LDL

The LDL was prepared as described earlier. The extent of LDL oxidationwas estimated by assaying for thiobarbituric acid reactive substances(TBARS) and expressed as nanomoles of malondialdehyde per mg of LDLprotein, as described earlier. Only freshly-isolated LDL with TBARSvalues of less than 0.5 nmol/mg was used in this study.

Western Blotting

Proteins were prepared and separated on SDS/PAGE as described earlier.Immuno-blotting was performed using monoclonal antibodies to ecNOS(1:400 dilution, Transduction Laboratories, Lexington, Ky.), to RhoA andRhoB (1:250 dilution, Santa Cruz Biotechnology Inc., Santa Cruz,Calif.), and to c-myc-tag (9E10, 1:200 dilution, Santa CruzBiotechnology Inc.). Immunodetection was accomplished using a sheepanti-mouse secondary antibody (1:4000 dilution) or donkey anti-rabbitsecondary antibody (1:4000 dilution) and the enhanced chemiluminescence(ECL) kit (Amersham Corp., Arlington Heights, Ill.). Autoradiography wasperformed as described earlier.

Assay for Rho GTP-binding Activity

The Rho GTP-binding activity was determined by immunoprecipitation of[³⁵ S]GTP S-labeled Rho. Briefly, membrane and cytosolic proteins wereisolated as previously desribed (Liao, J K and Homcy, C J, J ClinInvest, 1993, 92:2168-2172). Proteins (20 mg) from control and treatedendothelial cells were incubated for 30 min at 37° C. in a buffercontaining [35 S]GTP S (20 nM), GTP (2 mM), MgCl₂₂ (5 mM), EGTA (0.1mM), NaCl (50 mM), creatinine phosphate (4 mM), phosphocreatinine kinase(5 units), ATP (0.1 mM), dithiothreitol (1 mM), leupeptin (100 mg/ml),aprotinin (50 mg/ml), and phenylmethanesulfonyl fluoride (PMSF, 2 mM).The assay was terminated with excess unlabeled GTP S (100 mM).

Samples were then resuspended in 100 ml of immunopricipitation buffercontaining Triton-X (1%), SDS (0.1%), NaCl (150 mM), EDTA (5 mM),Tris-HCl (25 mM, pH 7.4), leupeptin (10 mg/ml), aprotinin (10 mg/ml),and PMSF (2 mM). The RhoA or RhoB antisera were added to the mixture ata final dilution of 1:75. The samples were allowed to incubate for 16 hat 4° C. with gentle mixing. The antibody-G-protein complexes were thenincubated with 50 ml of protein A-Sepharose (1 mg/ml, Pharmacia BiotechInc.) for 2 h at 4° C., and the immuno-precipitate was collected bycentrifugation at 12,000× g for 10 min.

Preliminary studies using Western analysis of the supernatant indicatedthat both RhoA and RhoB were completely immunoprecipitated under theseconditions. The pellets were washed four times in a buffer containingHEPES (50 mM, pH 7.4), NaF (100 mM), sodium phosphate (50 mM), NaCl (100mM), Triton X-100 (1%), and SDS (0.1%). The final pellet containing theimmunoprecipitated [35 S]GTP S-labeled Rho proteins was counted in aliquid scintillation counter (LS 1800, Beckman Instruments, Inc.Fullerton, Calif.). Nonspecific activity was determined in the presenceof excess unlabeled GTP S (100 mM).

Overexpression of Rho Mutants

For transfection studies, bovine rather than human endothelial cellswere used because of their higher transfection efficiency by thecalcium-phosphate precipitation method (12% vs <4%) (15). Bovineendothelial cells (60-70% confluent) were transfected with 15 mg of theindicated cDNAs: the insertless vector (pcDNA3), pcDNA3-c-myc-wtRhoA(wildtype RhoA), and pcDNA3-c-myc-N 19RhoA (dominant-negative RhoAmutant) (Gebbink, M et al., J Cell Biol, 1997, 137:1603-1613). As aninternal control for transfection efficiency, pCMV. b-Gal plasmid (5 mg)was co-transfected. Preliminary results using b-galactosidase stainingindicate that cellular transfection efficiency was approximately 10% to14%. The b-galactosidase activity was determined by a chemiluminescenceassay (Dual-Light, Tropix, Bedford, Mass.) using a Berthold L9501luminometer. Approximately 24 h after transfection, cells were harvestedfor immunoblot analyses of ecNOS expression. The ecNOS protein levelswere then standardized to the corresponding levels of transfected RhoAexpression as determined by antisera to the corresponding c-myc tag.

Assay for ecNOS Activity

The ecNOS activity was determined by a modified nitrite assay aspreviously described. Briefly, endothelial cells grown in phenol-freemedium were exposed to C3 transferase (50 mg/ml), FPP (10 mM), GGPP (5mM), CNF-1 (200 ng/ml), or mevastatin (10 mM). After 24 h, conditionedmedium (300 ml) was mixed with 30 ml of freshly-prepared2,3-diaminonaphthalene (1.5 mM of DAN in 1 M of HCI). The mixture wasprotected from light and incubated at 20° C. for 10 min. The reactionwas terminated with 15 ml of 2.8 M of NaOH. Fluorescence of1-(H)-naphthotriazole was measured with excitation and emissionwavelengths of 365 and 450 nm, respectively. Standard curves wereconstructed with known amounts of sodium nitrite. Nonspecificfluorescence was determined in the presence of LNMA (3 mM). Previousstudies with nitrate reductase indicate that the nitrite to nitrateconcentration in the medium was approximately 5:1 and that this ratiodid not vary under the described treatment conditions (Laufs, U et al.,J Biol Chem, 1997, 272:31725-31729).

Data Analysis

Band intensities from Northern and Western blots were analyzed asdescribed earlier. Paired and unpaired Student's t-tests were employedto determine the significance of changes in densitometric measurements,GTP-binding activities, and nitrite levels. A significant difference wastaken for p<0.05.

EXAMPLE 19 Cell Culture

Relatively pure (>98%) human saphenous vein endothelial cell cultureswere confirmed by their morphological features (i.e. cuboidal,cobble-stone, contact inhibited) using phase-contrast microscopy andimmunofluorescent-staining with antibodies to Factor VIII (data notshown). There were no observable adverse effects of mevastatin, FPP,GGPP, C3 transferase, and CNF-1 on cellular viability. However, higherconcentrations of mevastatin (>50 mM) or CNF-1 (>5 mg/ml) did producecytotoxicity and therefore were not used. Cellular confluency andviability as determined by light microscopy and trypan blue exclusionwere maintained for all treatment conditions described.

EXAMPLE 20 Effects of Isoprenoid Intermediates on ecNOS mRNA Expression

We have shown that inhibition of endothelial HMG-CoA reductase bylovastatin or simvastatin upregulates ecNOS expression and activity viaincreases in ecNOS mRNA stability (Examples 3-8 and Laufs, U et al., JBiol Chem, 1997, 272:31725-31729). Similarly, treatment of endothelialcells with mevastatin (10 mM) increased ecNOS steady-state mRNA levelsby 405±15% after 24 h (FIG. 3A). On a molar basis, we find thatmevastatin is equally potent compared with lovastatin but approximatelyten times less potent compared to simvastatin. This is consistent withtheir relative IC₅₀ values for HMG-CoA reductase inhibition (Blum, CB,Am. J. Cardiol, 1994, 73:3D-11D).

To determine which downstream isoprenoid intermediate in the cholesterolbiosynthetic pathway regulates ecNOS expression, endothelial cells weretreated with mevastatin (10 mM) in the presence or absence of isoprenoidintermediates, geranylgeranylpyrophosphate (GGPP) orfarnesylpyrophosphate (FPP). Co-treatment with FPP (10 mM) mildlyreduced ecNOS mRNA levels compared to mevastatin alone. However,co-treatment with GGPP (10 mM) completely reversed the upregulation ofecNOS mRNA levels by mevastatin. In a concentration-dependent manner,GGPP reversed the effects of mevastatin (10 mM) with complete reversaloccuring at a GGPP concentration of 5 mM (FIG. 3B). Interestingly,treatment with GGPP (10 mM) alone did not significantly affect basalecNOS mRNA levels.

Similarly, treatment with mevastatin (10 mM) increased ecNOS proteinlevels by 180±11% after 24 h (p<0.05, n=4) (FIG. 4). Co-treatment withFPP (10 mM) or LDL (1 mg/ml) did not significantly reverse the effectsof mevastatin on ecNOS protein levels. Furthermore, inhibition ofprotein farnesyltransferase with the farnesyl-protein transferaseinhibitor I (0.5-50 mM) or -hydroxyfarnesylphosphonic acid (2-20 mM) didnot affect ecNOS protein levels. In contrast, co-treatment with GGPP ata concentration of 10 mM, but not 1 mM, completely reversed theupregulation of ecNOS protein levels by mevastatin. These findingsindicate that ecNOS expression is negatively regulated bygeranylgeraniol synthesis.

EXAMPLE 21 Effects of Mevastatin on Rho Membrane Translocation

The geranylgeranylation of the small GTPases, RhoA and RhoB, areessential for their membrane translocation from the cytosol (Van Aelst,L and D'Souza-Schorey, C, Genes Dev, 1997, 11:2295-2322). Under basalculture conditions, both RhoA and RhoB are present in the membranes andcytosol. Treatment with mevastatin decreased membrane localization ofRhoA and RhoB by 60±5% and 78±6% and produce a concomitant increase inRhoA and RhoB in the cytosol by 65±4% and 87±7%. Co-treatment with GGPP(5 mM), but not FPP (10 mM) reversed the effects of mevastatin andcompletely restored the amount of cytosolic and membrane-asociated RhoAand RhoB to basal levels. These findings suggest that inhibition of Rhogeranylgeranylation by mevastatin prevents RhoA and RhoB fromtranslocating to and associating with the cellular membrane.

EXAMPLE 22 Effects of Mevastatin on Rho GTP-Binding Activity

To determine whether geranylgeranylation of RhoA and RhoB affects theiractivity (i.e. GTP-bound state), we immunoprecipitated [³⁵S]GTPS-labeled RhoA and RhoB from the membrane and cytosol of endothelialcells treated with mevastatin (10 mM) in the presence of GGPP (5 mM) orFPP (10 mM). Under basal conditions, endothelial cells havemembrane-associated RhoA and RhoB activity of 4.4±0.1 fmol/mg/min and3.8±0.4 fmol/mg/min, respectively. Treatment with mevastatin decreasedmembrane-associated RhoA and RhoB GTP-binding activity by 52% (2.1±0.4fmol/mg/min; p<0.01) and 37% (2.4±0.6 fmol/mg/min; p<0.05), respectively(n=3).

Co-treatment with FPP (10 mM) produced no significant effects on RhoAand RhoB GTP-binding activity compared to mevastatin alone (2.6±0.9fmol/mg/min and 2.7±0.5 fmol/mg/min, respectively, p>0.05, n=3).However, co-treatment with GGPP (10 mM) completely reversed theinhibitory effects of mevastatin on RhoA and RhoB GTP-binding activity(4.1±0.3 fmol/mg/min and 3.6±0.5 fmol/mg/min, respectively, p<0.05,n=3). Cytosolic RhoA and RhoB were relatively inactive (i.e. <1fmol/mg/min) and their activities were not affected by treatment withmevastatin alone or in combination with GGPP or FPP. Taken together,these results indicate that geranylgeranylation of RhoA and RhoB isnecessary for their membrane translocation and that membrane-associatedRho is relatively more active in terms of GTP-binding than cytosolicRho.

EXAMPLE 23 Effects of C3 Transferase on ecNOS Expression

To determine whether the inhibition of Rho mediates the effects ofmevastatin on ecNOS expression, endothelial cells were treated withmevastatin in the presence and absence of Clostridium botulinum C3transferase (5-50 mg/ml), an exoenzyme which specifically inactivatesRho by ADP-ribosylation (Aktories, K, J Clin Invest, 1997, 12:S11-S13).Treatment of endothelial cells with mevastatin (10 mM) or C3 transferase(50 mg/ml) for 48 h augmented ecNOS protein levels by 260±9% and250±10%, respectively (p<0.01, n=3). Lower concentrations of C3transferase (i.e. <50 mg/ml) produced correspondingly smaller increasesin ecNOS expression (data not shown). In contrast to the effect ofmevastatin, the stimulatory effect of C3 transferase on ecNOS expressionwas not reversed in the presence of L-mevalonate (200 mM).

EXAMPLE 24 Effects of Dominant-Negative RhoA on ecNOS Expression

Bovine aortic endothelial cells were transfected with insertless pcDNA3vector, c-myc-tagged wildtype RhoA (wtRhoA), or c-myc-taggeddominant-negative RhoA mutant (N19RhoA) which cannot exchange GDP forGTP and therefore is inactive (Gebbink, M et al., J Cell Biol, 1997,137:1603-1613). Immunostaining for b-galactosidase activity demonstratecomparable transfection efficiency of approximately 10% among the RhoAconstructs and between treatment conditions. To distinguish betweentransfected and endogenous RhoA, the amount of transfected RhoAconstructs expressed was assessed by immunoblotting using an antibody toc-myc (9E 10), which recognizes a 21 kD band only in wtRhoA and N19RhoAtransfected cells (FIG. 6).

Overexpression of wtRhoA mildly reduced basal ecNOS protein expressionby 15±4% suggesting that increased RhoA expression results in a decreasein basal ecNOS expression (p<0.05, n=3). Endothelial cells transfectedwith the dominant-negative N19RhoA mutant to comparable levels as wtRhoAas assessed by the amount of c-myc-tag, however, exhibited a 150±5%increase in ecNOS protein levels (p<0.05, n=3). The observed effects ofN19RhoA overexpression on overall ecNOS protein levels (i.e. transfectedand non-transfected cells) are more profound when one considers thatonly 10% of the endothelial cells were actually transfected. Thesefindings are consistent with our earlier findings that inhibition of RhoGTPase activity leads to an increase in ecNOS expression.

EXAMPLE 25 Effects of CNF-1 on ecNOS Expression

The Escherichia coli cytotoxic necrotizing factor (CNF)-1 is known todirectly and specifically activate rho proteins via glutaminedeamination (Aktories, K, J Clin Invest, 1997, 12:S11-S13; Schmidt, G etal., Nature, 1997, 387:725-729; Flatau, G et al., Nature, 1997,387:729-733). Treatment of endothelial cells with mevastatin (10 mM)increased ecNOS mRNA levels by 390±15% compared to basal levels (p<0.01,n=3). Co-treatment with CNF-1 (200 ng/ml) completely reversed theupregulation of ecNOS mRNA by mevastatin (p>0.05 compared to basallevels, n=3). Treatment with CNF-1 (200 ng/ml) alone, however, decreasedecNOS steady-state mRNA levels to 48±6% of basal levels at 24 h (p<0.05,n=3). These findings indicate that the direct activation of Rho leads tothe downregulation of ecNOS expression.

EXAMPLE 26 Effects of HMG-CoA Reductase Inhibitors and Rho on ecNOSActivity

The ecNOS activity was assessed by measuring the LNMA-inhibitablenitrite accumulation in conditioned media of endothelial cells (Laufs, Uet al., J Biol Chem, 1997, 272:31725-31729). Basal ecNOS activity was9.7±1.4 nmol/500,000 cells/24 hours (FIG. 7). Treatment of endothelialcells with mevastatin (10 mM) resulted in a 3-fold increase in nitriteaccumulation (32±1.9 nmol/500,000 cells/24 hours, p<0.01). This increasein ecNOS activity by mevastatin was reversed by co-treatment with GGPP(5 mM), but not FPP (10 mM) (12±0.8 and 27±4.9 nmol/500,000 cells/24hours, respectively). Furthermore, direct activation of Rho by CNF-1(200 ng/ml) reversed mevastatin-induced increase in ecNOS activity(32±1.9 to 14±2.1 nmol/500,000 cells/24 hours, p<0.05). In contrast,inhibition of Rho by C3 transferase (50 mg/ml) resulted in a 3-foldincrease in nitrite accumulation (31±2.1 and ±nmol/500,000 cells/24hours, p<0.05). These results indicate that Rho not only negativelyregulates ecNOS expression, but also ecNOS activity.

“Regulation of Endothelial Nitric Oxide Synthase Activity by Agents thatDisrupt Actin Cytoskeletal Organization”

Experimental Procedures

Materials:

Myosin light chain kinase inhibitors BDM [2,3-butanedione 2-monoxime],ML-7 [1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride], and H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydro-chloride], were purchased from Sigma Chemical Corp. (St. Louis,Mo.). Nocodazole

{Methyl-(5-[2-thienylcarbonyl]-1H-benzimidazol-2-yl)carbamate} was alsopurchased from Sigma.

Cell culture, Western blotting and Northern blotting were performed asdescribed earlier.

EXAMPLE 27 Effects of Agents That Disrupt Actin CytoskeletalOrganization on ecNOS Protein and mRNA Expression

To determine whether downstream targets of rhoGTPase exert any effect onecNOS expression, endothelial cells were treated in the presence andabsence of a myosin light chain (MLC) kinase inhibitor, for example,H-7. MLC kinase inhibitors decrease MLC phosphorylation and stress fiberformation. Treatment of endothelial cells with H-7 (1-100 mM) for 24hours augmented ecNOS protein levels (FIG. 8). Similar experiments usinga different MLC kinase inhibitor (ML-7), produced identical results.

Furthermore, disruption of the actin cytoskeletal organization byCytochalasin D (an agent that interferes with actin polymerization) orBDM (a different MLC kinase inhibitor), also lead to upregulation ofecNOS (FIGS. 9 and 10 respectively).

Inhibition, however, of cell cycle progression by anaphicoline (data notshown) or disruption of the microtubular cytoskeleton by enhancingmicrotubule depolymerization with nocodazole, do not increase ecNOSexpression (FIG. 11).

These findings show that the uregulation of ecNOS expression isrelatively specific to disruption of the actin cytoskeleton.Additionally, the increased ecNOS expression by Cytochalasin D or BDMdoes not result from nonspecific cytotoxicity of these agents.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. A) NOS-Activity measured by (C14)-arginine-citrulline assay inthe aortas of wild-type SV-129 mice after treatment with simvastatin(Sim, 2 mg/kg, s.c., 14 days) and of mice injected with PBS (Control),n=8, p<0.05.

B) ecNOS mRNA expression determined by quantitative polymerase chainreaction in wild-type SV-129 mice aortas after treatment withsimvastatin (Sim0.2, 0.2 mg/kg, s.c. and Sim20, 20 mg/kg, s.c.) for 14days and of mice injected with saline (Control) in comparison toglyceraldehyde 3-phosphate dehydrogenase (G3DPH) mRNA expression.

ecNOS expression and function is upregulated in the aortas of micetreated with Sim.

FIG. 2. ecNOS mRNA expression in the infarcted, ipsolateral (I) andnot-infarcted, contralateral (C) forebrain hemispheres of SV-129 miceafter treatment with simvastatin (Sim, 2 mg/kg, s.c., 14 days) and miceinjected with saline (Control), as determined by quantitative polymerasechain reaction compared to glyceraldehyde 3-phosphate dehydrogenase(G3DPH) mRNA expression. ecNOS mRNA expression was upregulated in theinfarcted brain area in Sim-treated animals.

FIG. 3. A) Northern analyses (20 mg total RNA/lane) showing the effectsof mevastatin (Statin, 10 mM) alone or in combination with FPP (10 mM)or GGPP (10 mM) on eNOS steady-state mRNA levels at 24 h. B)Concentration-dependent effects of GGPP (1-10 mM) on mevastatin (10mM)-induced increases in eNOS mRNA levels after 24 h. Each experimentwas performed three times with comparable results. The correspondingethidium bromide-stained 28S band intensities were used to standardizeloading conditions.

FIG. 4. Immunoblots (30 mg protein/lane) showing the effects ofmevastatin (Statin, 10 mM) alone or in combination with FPP (10 mM),GGPP (1-10 mM), or LDL cholesterol (LDL-C, 1 mg/ml) on eNOS proteinlevels after 24 h. The blot is representative of three separateexperiments.

FIG. 5. Immunoblot (30 mg protein/lane) showing the effects of C3transferase (C3, 50 mg/ml), mevastatin (Statin, 10 mM), or L-mevalonate(Mev, 200 mM) on eNOS protein levels after 48 h. The blot isrepresentative of three separate experiments.

FIG. 6. Immunoblots (30 mg protein/lane) showing eNOS protein levelsafter transfection with insertless vector, pcDNA3 (C),c-myc-wildtype-RhoA (wt), and c-myc-N19RhoA (dominant-negative rhoAmutant). The levels of overexpressed RhoA mutants were determined byimmunoblotting for their corresponding c-myc-tags (c-myc-RhoA).Experiments were performed three times with similar results.

FIG. 7. Effects of C3 transferase (C3, 50 mg/ml), FPP (10 mM), GGPP (5mM), and CNF-1 (200 ng/ml) on mevastatin (Statin, 10 mM)-induced eNOSactivity as determined by LNMA-inhibitable nitrite production at 24 h.Experiments were performed three times in duplicate with less than 5%variation. *p<0.05 compared with control (C), **p<0.05 compared withmevastatin.

FIG. 8. Immunoblots (30 mg protein/lane) showing theconcentration-dependent effects of MLC kinase inhibitor H-7on ecNOSprotein levels after 24 hours.

FIG. 9. Northern blot analysis (20 mg total RNA/lane) showing ecNOS mRNAexpression of endothelial cells treated with cytochalasin D at 24 hours.

FIG. 10. Immunoblots (30 mg protein/lane) showing theconcentration-dependent effects of 2,3-butanedione 2-monoxime on ecNOSprotein levels.

FIG. 11. Northern blot analysis (20 mg total RNA/lane) showing ecNOSmRNA expression of endothelial cells treated with nocodazole for 24hours.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

A Sequence Listing follows the claims.

All references disclosed herein are incorporated by reference in theirentirety.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 4 <210> SEQ ID NO 1 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 1gggctccctc cttccggctg ccacc           #                  #               25 <210> SEQ ID NO 2 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 2ggatccctgg aaaaggcggt gagg           #                  #                24 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 3accacagtcc atgccatcac             #                  #                   # 20 <210> SEQ ID NO 4 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 4tccaccaccc tgttgctgta             #                  #                   # 20

I claim:
 1. A method for treating a hypoxia-induced condition in asubject, comprising: administering to a subject who has an abnormallyelevated risk of developing a hypoxia-induced condition or who has ahypoxia-induced condition an agent that disrupts actin cytoskeletalorganization in an amount effective to increase endothelial cell NitricOxide Synthase activity in the subject, provided that the agent thatdisrupts actin cytoskeletal organization is not a rho GTPase functioninhibitor.
 2. The method of claim 1, wherein the subject isnonhyperlipidemic.
 3. The method of claim 1, wherein the agent thatdisrupts actin cytoskeletal organization is administeredprophylactically to a subject who has an abnormally elevated risk ofdeveloping a hypoxia-induced condition.
 4. The method of claim 1,wherein the agent that disrupts actin cytoskeletal organization isadministered to a subject who has a hypoxia-induced condition.
 5. Themethod of claim 1, wherein the subject has an impaired lung function. 6.The method according to any one of claims 1-5, wherein the agent thatdisrupts actin cytoskeletal organization is selected from the groupconsisting of a myosin light chain kinase inhibitor, a myosin lightchain phosphatase, a protein kinase N inhibitor, a phospatidylinositol4-phosphate 5-kinase inhibitor, and cytochalasin D.
 7. The method ofclaim 6, wherein the myosin light chain kinase inhibitor is selectedfrom the group consisting of 2,3-butanedione 2-monoxime,1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepinehydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydro-chloride.
 8. The method according to any one of claims 1-5,further comprising co-administering a substrate of endothelial cellNitric Oxide Synthase.
 9. The method according to any one of claims 1-5,further comprising co-administering an agent other than an agent thatdisrupts actin cytoskeletal organization that increases endothelial cellNitric Oxide Synthase activity.
 10. The method according to any one ofclaims 1-5, further comprising co-administering at least one differentagent that disrupts actin cytoskeletal organization in an amounteffective to increase endothelial cell Nitric Oxide Synthase activity insaid tissue of the subject.